Variants of hepatitis B virus with resistance to anti-viral nucleoside agents and applications thereof

ABSTRACT

The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to these antibodies. The present invention further contemplates assays for detecting such viral variants, which assays are useful in monitoring anti-viral therapeutic regimens and in developing new or modified vaccines directed against viral agents and in particular HBV variants. The present invention also contemplates the use of the viral variants to screen for and/or develop or design agents capable of inhibiting infection, replication and/or release of the virus.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/978,289, filed Dec. 23, 2010, which is a continuation of U.S. patentapplication Ser. No. 11/932,410, filed Oct. 31, 2007, which is acontinuation of U.S. patent application Ser. No. 11/911,097 filed Oct.9, 2007 which is the U.S., National Phase under 35 U.S.C. §371 ofInternational Application PCT/AU2006/000450, filed Apr. 4, 2006designating the U.S., and published in English as WO 2006/105597 on Oct.12, 2006, which claims priority to Australian Patent Application No.2005901757 filed Apr. 8, 2005 and Australian Patent Application No.2005903972 filed Jul. 26, 2005, all of which are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to viral variants exhibitingreduced sensitivity to particular agents and/or reduced interactivitywith immunological reagents. More particularly, the present invention isdirected to hepatitis B virus (HBV) variants exhibiting complete orpartial resistance to nucleoside or nucleotide analogs and/or reducedinteractivity with antibodies to viral surface components includingreduced sensitivity to these antibodies. The present invention furthercontemplates assays for detecting such viral variants, which assays areuseful in monitoring anti-viral therapeutic regimens and in developingnew or modified vaccines directed against viral agents and in particularHBV variants. The present invention also contemplates the use of theviral variants to screen for and/or develop or design agents capable ofinhibiting infection, replication and/or release of the virus.

DESCRIPTION OF THE PRIOR ART

Bibliographic details of the publications referred to in thisspecification are also collected at the end of the description.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in any country.

Hepatitis B virus (HBV) can cause debilitating disease conditions andcan lead to acute liver failure. HBV is a DNA virus which replicates viaan RNA intermediate and utilizes reverse transcription in itsreplication strategy (Summers and Mason, Cell 29: 403-415, 1982). TheHBV genome is of a complex nature having a partially double-stranded DNAstructure with overlapping open reading frames encoding surface, core,polymerase and X genes. The complex nature of the HBV genome isrepresented in FIG. 1. The polymerase consists of four functionalregions, the terminal protein (TP), spacer, reverse transcriptase (rt)and ribonuclease (RNAse).

The polymerase gene of HBV overlaps the envelope gene, mutations in thecatalytic domain of the polymerase gene can also affect the nucleotideand the deduced amino acid sequence of the envelope protein and viceversa. In particular, the genetic sequence for the neutralization domainof HBV known as the ‘a’ determinant, which is found within the HBsAg andlocated between amino acids 99 and 169, actually overlaps the majorcatalytic regions of the viral polymerase protein and in particulardomains A and B.

The presence of an HBV DNA polymerase has led to the proposition thatnucleoside or nucleotide analogs could act as effective anti-viralagents. Examples of nucleoside or nucleotide analogs currently beingtested are penciclovir and its oral form (FCV) [Vere Hodge, AntiviralChem Chemother 4: 67-84, 1993; Boyd et al., Antiviral Chem Chemother.32: 358-363, 1987; Kruger et al., Hepatology 22: 219A, 1994; Main etal., J. Viral Hepatitis 3: 211-215, 1996], Lamivudine[(−)-β-2′-deoxy-3′-thiacytidine]; (3TC or LMV) [Severini et al.,Antimicrobial Agents Chemother. 39: 430-435, 1995; Dienstag et al., NewEngland J Med 333: 1657-1661, 1995]. New nucleoside or nucleotideanalogs which have already progressed to clinical trials include thepyrimidines Emtricitabine,((−)-β-L-2′-3′-dideoxy-5-fluoro-3′-thiacydidine; FTC), the 5-fluoroderivative of 3TC, and Clevudine(1-(2-fluoro-5-methyl-β-L-arabino-furanosyl) uracil; L-FMAU), athymidine analog. Like 3TC, these are pyrimidine derivatives with anunnatural “L”-configuration. Several purine derivatives have alsoprogressed to clinical trials; they include Entecavir (BMS-200, 475;ETV), a carbocyclic deoxyguanosine analog, diaminopurine dioxolane(DAPD), an oral pro-drug for dioxolane guanine ((−)-β-D-2-aminopurinedioxolane; DXG) and Adefovir dipivoxil, an oral prodrug for the acyclicdeoxyadenosine monophosphate nucleoside or nucleotide analog Adefovir(9-[phosphonyl-methoxyethyl]-adenine; PMEA). Other drugs in pre-clinicaland clinical trials include FLG [Medivir], ACH-126,443 (L-d4C)[Archillion Pharmaceuticals], ICN 2001-3 (ICN) and Racivir (RCV)[Pharmassett].

Whilst these agents are highly effective in inhibiting HBV DNAsynthesis, there is the potential for resistant mutants of HBV to emergeduring long term antiviral chemotherapy. In patients on prolonged LMVtherapy, key resistance mutations are selected in the rt domain withinthe polymerase at rtM204I/V+/−rtL180M as well as other mutations. Thenomenclature used for the polymerase mutations is in accordance withthat proposed by Stuyver et al., 2001, supra. LMV is a nucleoside oranucleotide analog that has been approved for use against chronic HBVinfection. LMV is a particularly potent inhibitor of HBV replication andreduces HBV DNA titres in the sera of chronically infected patientsafter orthotopic liver transplantation (OLT) by inhibiting viral DNAsynthesis. LMV monotherapy seems unlikely to be able to control HBVreplication in the longer term. This is because emergence ofLMV-resistant strains of HBV seems almost inevitable during monotherapy.

Adefovir dipivoxil (ADV: formerly, bis-pom PMEA) is an orally availableprodrug of the acyclic deoxyadenosine monophosphate analog adefovir(formerly, PMEA) (FIG. 2). ADV is also a potent inhibitor of HBVreplication and has recently been given FDA approval for use againstchronic HBV infection. Adefovir dipivoxil differs from other agents inthis class in that it is a nucleotide (vs. nucleoside) analog and assuch bypasses the first phosphorylation reaction during drug activation.This step is often rate-limiting. Adefovir dipivoxil has demonstratedclinical activity against both wild-type and lamivudine-resistantstrains of HBV and is currently in phase III clinical Testing (Gilson etal., J Viral Hepat 6: 387-395, 1999; Perrillo et al., Hepatology 32:129-134, 2000; Peters et al., Transplantation 68: 1912-1914, 1999;Benhamou et al., Lancet 358: 718-723, 2001). During phase II studies a30 mg daily dose of adefovir dipivoxil resulted in a mean 4 log₁₀decrease in viremia over 12 weeks (Heathcote et al., Hepatology 28:A620, 1998).

ADV is a substituted acyclic nucleoside phosphonate. This class ofcompounds also includes tenofovir disoproxil fumarate (also referred toas tenofovir DF, or tenofovir, or (TFV) or9-R-(2-phosphonomethoxypropyl)adenine (PMPA) and is marketed as Vireadby Gilead sciences).

TFV has antiviral activity against both HBV and HIV (Ying et al., JViral Hepat. 7(2): 161-165, 2000; Ying et al., J. Viral Hepat. 7(1):79-83, 2000, 2000; Suo et al., J Biol Chem. 273(42): 27250-27258. 1998).

FTC has activity against HBV and HIV (Frick et al., Antimicrob AgentsChemother 37: 2285-2292, 1993).

Nucleoside or nucleotide analog therapy may be administered asmonotherapy or combination therapy where two or more nucleoside ornucleotide analogs may be administered. The nucleoside or nucleotideanalogs may also be administered in combination with other antiviralagents such as interferon or hepatitis B immunoglobulin (HBIG).

There is a need to monitor for the emergence ofnucleoside/nucleotide-analog- or antibody-resistant strains of HBV andto develop diagnostic protocols to detect these resistant viruses and/orto use them to screen for and/or develop or design agents havingproperties making them useful as anti-viral agents. Defective forms ofthese resistant strains or antigenic components therefrom are alsoproposed to be useful in the development of therapeutic vaccinecompositions as are antibodies directed to viral surface components.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

Specific mutations in an amino acid sequence are represented herein as“Xaa₁nXaa₂” where Xaa₁ is the original amino acid residue beforemutation, n is the residue number and Xaa₂ is the mutant amino acid. Theabbreviation “Xaa” may be the three letter or single letter (i.e. “X”)code. An “rt” before “Xaa₁nXaa₂” means “reverse transcriptase”. An “s”means an envelope gene. The amino acid residues for HBV DNA polymeraseare numbered with the residue methionine in the motif Tyr Met Asp Asp(YMDD) being residue number 204 (Stuyver et al., Hepatology 33: 751-757,2001). The amino acid residues for hepatitis B virus surface antigen arenumber according to Norder et al. (J. Gen. Virol. 74: 341-1348, 1993).Both single and three letter abbreviations are used to define amino acidresidues and these are summarized in Table 2.

In accordance with the present invention, the selection of HBV variantsis identified in patients with chronic HBV infection treated with ADVand/or LMV. Consequently, HBV rt variants are contemplated which areresistant to, or which exhibit reduced sensitivity to, ADV, LMV, TFV orFTC; ADV and LMV; ADV and TFV; FTC and ADV; FTC and TFV; FTC and LMV;ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADVand FTC and LMV and TFV and ETV and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combinations thereofand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof. Corresponding mutations in thesurface antigen also occur. The identification of these HBV variants isimportant for the development of assays to monitor ADV, LMV, FTC and/orTFV resistance and/or resistance with ETV in combination with anynucleoside or nucleotide analog with the exception of LMV and/orresistance to other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof and to screen for agents which are usefulas alternative therapeutic agents. The subject variants are not proposedto exhibit resistance to ETV alone or ETV and LMV alone.

Reference herein to “anti-HBV agents” includes nucleoside and nucleotideanalogs as well as immunological reagents (e.g. antibodies to HBVsurface components) and chemical, proteinaceous and nucleic acid agentswhich inhibit or otherwise interfere with viral replication,maintenance, infection, assembly or release.

The detection of such HBV variants is particularly important in themanagement of therapeutic protocols including the selection ofappropriate agents for treating HBV infection. The method of this aspectof the present invention is predicated in part on monitoring thedevelopment in a subject of an increased HBV load in the presence of anucleoside or nucleotide analog or other anti-HBV agents or combinationsthereof. The clinician is then able to modify an existing treatmentprotocol or select an appropriate treatment protocol accordingly.

Accordingly, one aspect of the present invention is directed to anisolated Hepatitis B virus (HBV) variant wherein said variant comprisesa nucleotide mutation in a gene encoding a DNA polymerase resulting inat least one amino acid addition, substitution and/or deletion to saidDNA polymerase and wherein said variant exhibits decreased sensitivityto one or more nucleoside or nucleotide analogs selected from the listconsisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV andTFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC;ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTCand LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV;ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMVand TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADVand FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof. With the proviso that the HBV variantdoes not exhibit resistance to ETV alone or ETV or LMV alone. Thevariant HBV comprises a mutation in an overlapping open reading frame inits genome in a region defined by one or more of domains F and G anddomain A through to E of HBV DNA polymerase.

Another aspect of the present invention provides an isolated HBV variantcomprising a nucleotide mutation in the S gene resulting in at least oneamino acid addition, substitution and/or deletion to the surface antigenand which exhibits decreased sensitivity to one or more nucleoside ornucleotide analogs selected from the list consisting of ADV, LMV, TFV orFTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV;FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADVand FTC and LMV and TFV and ETV and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combinations thereof.With the proviso that the HBV variant does not exhibit resistance to ETValone or ETV or LMV alone.

Useful mutants in the rt region include, in one embodiment, mutations atcodons rtN53, rtY54, rtL180, rtT181, rtT184, rtM204, rtN236, rtY124,rtH126, rtT128, rtS135, rtL180, rtS202, rtM204, rtH248, rtI53, rtS54,rtN122, rtM145, rtL180, rtM204, rtM250, rtN53, rtS85, rtS116, rtD134,rtN139, rtQ149, rtA181, rtS219, rtI233, rtN236 or rtM250. Particularmutants include rtN53, rtN53, rtY54, rtL180, rtT181, rtT184, rtM204, andrtN236, in another embodiment include rtY124, rtH126, rtT128, rtS135,rtL180, rtS202, rtM204, and rtH248, or yet another embodiment includertI53, rtS54, rtN122, rtM145, rtL180, rtM204 and rtM250, or yet anotherembodiment rtN53, rtS85, rtS116, rtD134, rtN139, rtQ149, rtA181, rtS219,rtI233, rtN236, rtM250 or a combination thereof or an equivalentmutation. Even more particular mutants include rtN53K, rtN53K/N, rtY54D,rtL180M, rtT181A/V, rtT184S, rtM204V, and rtN236T, in another embodimentinclude rtY124H, rtH126R, rtT128N, rtS135C, rtL180M rtS202C rtM204V, andrtH248N, or yet another embodiment include rtI53V, rtS54T, rtN122T,rtM145L, rtL180M, rtM204V and rtM250L, or yet another embodiment rtN53D,rtS85A, rtS116P, rtD134V, rtN139E/K, rtQ149K, rtA181V, rtS219A, rtI233V,rtN236T, rtM250L or a combination thereof or an equivalent mutation.

Particularly useful mutants are at codons rtT184, rtS202, rtS219,rtI233, rtH248 or rtM250 such as rtT184S, rtS202C, rtS219A, rtI233V,rtH248N or rtM250L.

Useful mutations in the S gene include, in one embodiment includemutations at codons stT45, sL173, sL175, sI195, sT118, sP120, sP127,sI195, sT114, SI195, sS204, sI208, sS210, sV14, sG130, sM133, sW172,sS204 or sS210 such as sT45K, sL173L/F, sL175F, sI195M, in anotherembodiment include sT118A, sP120T, sP127A and sI195M or yet anotherembodiment include sT114P, sI195M, sS204N, sI208T and sS210R and finallyanother embodiment include sV14A, sG130R, sM133T, sW172L, sS204G,sS210R.

The present invention further contemplates a method for determining thepotential for an HBV to exhibit reduced sensitivity to ADV, LMV, TFV orFTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV;FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADVand FTC and LMV and TFV and ETV and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combinations thereof byisolating DNA or corresponding mRNA from the HBV and screening for amutation in the nucleotide sequence encoding HBV DNA polymeraseresulting in at least one amino acid substitution, deletion and/oraddition in any one or more of domains F and G and domains A through toE or a region proximal thereto of the DNA polymerase and associated withresistance or decreased sensitivity to ADV, LMV, TFV or FTC; ADV andLMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV;ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV andFTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV andTFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV andTFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV andFTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMVand TFV and ETV and/or optionally other nucleoside or nucleotide analogsor other anti-HBV agents or combinations thereof. With the proviso thatthe HBV variant does not exhibit resistance to ETV alone or ETV or LMValone.

The presence of such a mutation is an indication of the likelihood ofresistance to ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV andTFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC;ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTCand LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV;ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMVand TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADVand FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof.

The present invention also provides a composition comprising a variantHBV resistant to ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV andTFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC;ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTCand LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV;ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMVand TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADVand FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof or an HBV surface antigen from thevariant HBV or a recombinant or derivative form thereof or its chemicalequivalent and one or more pharmaceutically acceptable carriers and/ordiluents. With the proviso that the HBV variant does not exhibitresistance to ETV alone or ETV or LMV alone.

Yet another aspect of the present invention provides a use of theaforementioned composition or a variant HBV comprising a nucleotidemutation in a gene encoding a DNA polymerase resulting in at least oneamino acid addition, substitution and/or deletion to the DNA polymeraseand a decreased sensitivity to ADV, LMV, TFV or FTC; ADV and LMV; ADVand TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV andADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC andTFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETVand LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETVand FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC andETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFVand ETV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combinations thereof in the manufacture of amedicament for the treatment and/or prophylaxis of hepatitis B virusinfection. With the proviso that the HBV variant does not exhibitresistance to ETV alone or ETV or LMV alone.

The present invention also contemplates a method for determining whetheran HBV strain exhibits reduced sensitivity to a nucleoside or nucleotideanalog or other anti-HBV agents or by isolating DNA or correspondingmRNA from the HBV and screening for a mutation in the nucleotidesequence encoding the DNA polymerase wherein the presence of thefollowing mutations in the rt region: in one embodiment, mutations atcodons rtN53, rtY54, rtL180, rtT181, rtT184, rtM204, rtN236, rtY124,rtH126, rtT128, rtS135, rtL180, rtS202, rtM204, rtH248, rtI53, rtS54,rtN122, rtM145, rtL180, rtM204, rtM250, rtN53, rtS85, rtS116, rtD134,rtN139, rtQ149, rtA181, rtS219, rtI233, rtN236 or rtM250. Particularmutants include rtN53, rtN53, rtY54, rtL180, rtT181, rtT184, rtM204, andrtN236, in another embodiment include rtY124, rtH126, rtT128, rtS135,rtL180, rtS202, rtM204, and rtH248, or yet another embodiment includertI53, rtS54, rtN122, rtM145, rtL180, rtM204 and rtM250, or yet anotherembodiment rtN53, rtS85, rtS116, rtD134, rtN139, rtQ149, rtA181, rtS219,rtI233, rtN236, rtM250 or a combination thereof or an equivalentmutation. Even more particular mutants include rtN53K, rtN53KIN, rtY54D,rtL180M, rtT181A/V, rtT184S, rtM204V, and rtN236T, in another embodimentinclude rtY124H, rtH126R, rtT128N, rtS135C, rtL180M rtS202C rtM204V, andrtH248N, or yet another embodiment include rtI53V, rtS54T, rtN122T,rtM145L, rtL180M, rtM204V and rtM250L, or yet another embodiment rtN53D,rtS85A, rtS116P, rtD134V, rtN139E/K, rtQ149K, rtA181V, rtS219A, rtI233V,rtN236T, rtM250L or a combination thereof or an equivalent mutation. Themutations may also be a combination thereof or an equivalent mutationwhich mutation(s) is/are indicative of a variant which exhibits adecreased sensitivity to ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV;LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV andFTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV andFTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV andTFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC andLMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV;ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof. With the proviso that the HBVvariant does not exhibit resistance to ETV alone or ETV or LMV alone.

Still a further aspect provides a method for determining whether an HBVstrain exhibits reduced sensitivity to a nucleoside or nucleotide analogsaid method comprising screening for a mutation in the nucleotidesequence encoding the envelope genes (s) wherein the presence of thefollowing mutations in the s gene: in one embodiment include mutationsat codons stT45, sL173, sL175, sI195, sT118, sP120, sP127, sI195, sT114,SI195, sS204, sI208, sS210, sV14, sG130, sM133, sW172, sS204 or sS210such as sT45K, sL173L/F, sL175F, sI195M and in another embodimentinclude and sT118A, sP120T, sP127A and sI195M, or yet another embodimentinclude sT114P, sI195M, sS204N, sI208T, and sS210R, or still yet anotherembodiment include sV14A, sG130R, sM133T, sW172L, sS204G, sS210R or acombination thereof or an equivalent mutation or combinations thereof oran equivalent one or more other mutation is indicative of a variantwhich exhibits a decreased sensitivity to ADV, LMV, TFV or FTC; ADV andLMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV;ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV andFTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV andTFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV andTFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV andFTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMVand TFV and ETV and/or optionally other nucleoside or nucleotide analogsor other anti-HBV agents or combinations thereof, and/or optionallyother nucleoside or nucleotide analogs or other anti-HBV agents orcombination thereof. With the proviso that the HBV variant does notexhibit resistance to ETV alone or ETV or LMV alone.

Preferably, the variants are in an isolated form such that they haveundergone at least one purification step away from naturally occurringbody fluid. Alternatively, the variants may be maintained in isolatedbody fluid or may be in DNA form. The present invention alsocontemplates infectious molecular clones comprising the genome or partsthereof from a variant HBV. The detection of HBV or its components incells, cell lysates, cultured supernatant fluid and bodily fluid may beby any convenient means including any nucleic acid-based detectionmeans, for example, by nucleic acid hybridization techniques or via oneor more polymerase chain reactions (PCRs). The term “bodily fluid”includes any fluid derived from the blood, lymph, tissue or organsystems including serum, whole blood, biopsy and biopsy fluid, organexplants and organ suspension such as liver suspensions.

Another aspect of the present invention is directed to a variant HBVcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion or atruncation compared to a surface antigen from a reference or wild typeHBV and wherein an antibody generated to the reference or wild typesurface antigen exhibits an altered immunological profile relative tothe HBV variant. One altered profile includes a reduced capacity forneutralizing the HBV. More particularly, the surface antigen of thevariant HBV exhibits an altered immunological profile compared to apre-treatment HBV where the variant HBV is selected for by a nucleosideor nucleotide analog or other anti-HBV agents of the HBV DNA polymerase.The variant HBV of this aspect of the invention may also comprise anucleotide sequence comprising a single or multiple nucleotidesubstitution, addition and/or deletion compared to a pre-treatment HBV.

The present invention further contemplates a method for detecting avariant HBV exhibiting an altered immunological profile said methodcomprising isolating an HBV from a subject exposed to a nucleoside ornucleotide analog or combination of analogs selected from the listedconsisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV andTFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC;ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTCand LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV;ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMVand TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADVand FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof, and then contacting said HBV with apanel of one or more antibodies to a surface antigen and screening forany change in binding affinity or binding spectrum. With the provisothat the HBV variant does not exhibit resistance to ETV alone or ETV orLMV alone.

In a related invention, the present invention provides a method fordetecting a variant HBV exhibiting an altered immunological profile saidmethod comprising isolating a serum sample from a subject exposed to anucleoside or nucleotide analog selected from the listed consisting ofADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC andADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV;ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADVand LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMVand FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADVand ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC andLMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionallyother nucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof, and then contacting the serum with a panel of HBVsurface antigens or antibody-binding fragments thereof and screening forany change in binding affinity or binding spectrum. With the provisothat the HBV variant does not exhibit resistance to ETV alone or ETV orLMV alone.

The present invention extends to an isolated HBsAg or a recombinant formthereof or derivative or chemical equivalent thereof corresponding tothe variant HBV. Generally, the HBsAg or its recombinant or derivativeform or its chemical equivalent comprises an amino acid sequence with asingle or multiple amino acid substitution, addition and/or deletion ora truncation compared to an HBsAg from a reference HBV and wherein anantibody directed to a reference HBV exhibits an altered immunologicalprofile to an HBV carrying said variant HBsAg. In one embodiment, thealtered immunological profile comprises a reduction in the ability toneutralize the variant HBV.

Another aspect of the present invention contemplates a method fordetecting an agent which exhibits inhibitory activity to an HBV bygenerating a genetic construct comprising a replicationcompetent-effective amount of the genome from the HBV contained in aplasmid vector and then transfecting said cells with said construct,contacting the cells, before, during and/or after transfection, with theagent to be tested, culturing the cells for a time and under conditionssufficient for the HBV to replicate, express genetic sequences and/orassemble and/or release virus or virus-like particles if resistant tosaid agents; and the subjecting the cells, cell lysates or culturesupernatant fluid to viral- or viral-component-detection means todetermine whether or not the virus has replicated, expressed geneticmaterial and/or assembled and/or been released in the presence of theagent. In a preferred embodiment, the plasmid vector in a baculovirusvector and the method comprises generating a genetic constructcomprising a replication competent-effective amount of the genome fromthe HBV contained in or fused to an amount of a baculovirus genomeeffective to infect cells and then infecting said cells with saidconstruct, contacting the cells, before, during and/or after infection,with the agent to be tested, culturing the cells for a time and underconditions sufficient for the HBV to replicate, express geneticsequences and/or assemble and/or release virus or virus-like particlesif resistant to said agent and then subjecting the cells, cell lysatesor culture supernatant fluid to viral- or viral-component-detectionmeans to determine whether or not the virus has replicated, expressedgenetic material and/or assembled and/or been released in the presenceof the agent.

In connection with these methods, the plasmid vector may include genesencoding part or all of other viral vectors such as baculovirus vectorsor adenovirus vectors (see Ren and Nassal, J. Virol. 75(3): 1104-1116,2001).

In an alternative embodiment, the method comprises generating acontinuous cell line comprising an infectious copy of the genome of theHBV in a replication competent effective amount such that saidinfectious HBV genome is stably integrated into said continuous cellline such as but not limited to the 2.2.15 or AD cell line, contactingthe cells with the agent to be tested, culturing the cells for a timeand under conditions sufficient for the HBV to replicate, expressgenetic sequences and/or assemble and/or release virus or virus-likeparticles if resistant to the agent and then subjecting the cells, celllysates or culture supernatant fluid to viral- orviral-component-detection means to determine whether or not the virushas replicated, expressed genetic material and/or assembled and/or beenreleased in the presence of the agent.

In an alternative embodiment, the present invention also contemplates amethod for detecting an agent which exhibits inhibitory activity to anHBV polymerase in an in vitro polymerase assay. The HBV polymeraseactivity can be examined using established assays (Gaillard et al.,Antimicrob Agents Chemother. 46(4): 1005-1013, 2002; Xiong et al.,Hepatology. 28(6): 1669-73, 1998). The HBV polymerase may be a wild-typeor reference HBV polymerase or mutant HBV polymerase.

The identification of viral variants enables the production of vaccinescomprising particular recombinant viral components such as polymerasesor envelope genes PreS1, PreS2, S encoding for L, M, S proteins as wellas therapeutic vaccines comprising defective HBV variants. Rational drugdesign may also be employed to identify or generate therapeuticmolecules capable of interacting with a polymerase or envelope genesPreS1, PreS2, S encoding for L, M, S proteins or other component of theHBV. Such drugs may also have diagnostic potential. In addition,defective HBV variants may also be used as therapeutic compositions togenerate an immune response against the same, similar or homologousviruses. Alternatively, antibodies generated to the HBV variants orsurface components thereof may be used in passive immunization ofsubjects against infection by HBV variants or similar or homologousviruses. Furthermore, agents such as nucleoside or nucleotide analogs,RNAi or siRNA molecules (both DNA-derived or synthetic), antisense orsense oligonucleotides, chemical or proteinaceous molecules having anability to down-regulate the activity of a component of HBV and inhibitreplication, maintenance, infection, assembly or release arecontemplated by the present invention.

As indicated above, the present invention does not extend to an HBVvariant exhibiting resistance or reduced sensitivity to ETV alone or ETVand LMV alone.

A summary of the abbreviations used throughout the subject specificationare provided in Table 3.

A summary of sequence identifiers used throughout the subjectspecification is provided in Table 1.

TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1PCR primer 2 PCR primer 3 PCR primer 4 PCR primer 5 PCR primer 6Nucleotide sequence of catalytic region of polymerase from resistant HBVPatient A: Sample A 7 Deduced amino acid sequence of catalytic region ofpolymerase resistant HBV Patient A: Sample A 8 Dedecuded amino acidsequence of envelope gene from resistant HBV from Pateint A: Sample A 9Nucleotide sequence of catalytic region of polymerase from resistant HBVPatient A: Sample B 10 Dedecuded amino acid sequence of envelope genefrom resistant HBV from Pateint A: Sample B 11 Dedecuded amino acidsequence of envelope gene from resistant HBV from Pateint A: Sample B 12Nucleotide sequence of catalytic region of polymerase from resistant HBVPatient B 13 Deduced amino acid sequence of catalytic region ofpolymerase resistant HBV Patient B 14 Dedecuded amino acid sequence ofenvelope gene from resistant HBV from Pateint B 15 Nucleotide sequenceof catalytic region of polymerase from resistant HBV Patient C 16Deduced amino acid sequence of catalytic region of polymerase resistantHBV Patient C 17 Dedecuded amino acid sequence of envelope gene fromresistant HBV from Pateint C 18 Nucleotide sequence of catalytic regionof polymerase from resistant HBV Patient D 19 Deduced amino acidsequence of catalytic region of polymerase resistant HBV Patient D 20Dedecuded amino acid sequence of envelope gene from resistant HBV fromPateint D

TABLE 2 Single and three letter amino acid abbreviations Amino AcidThree-letter Abbreviation One-letter symbol Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln QGlutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine The T Tryptophan Trp W Tyrosine Tyr YValine Val V Any residue Xaa X

TABLE 3 Abbreviations ABBREVIATION DESCRIPTION 3TC (LMV);(−)-β-2′-deoxy-3′- thiacytidine ADV adefovir dipivoxil DAPDdiaminopurine dioxalone DXG dioxolane guanine ETV entecavir FAMfamciclovir FCV famciclovir FTC emtricitabine HBIG hepatitis Bimmunoglobulin HBsAg hepatitis B surface antigen HBV hepatitis B virusLMV lamividuine PMEA 9-[phosphonyl-methoxyethyl]-adenine; adefovir PMPA9-R-(2-phosphonomethoxypropyl)adenine RNase ribonuclease rt (“rt” beforereverse transcriptase “Xaa₁nXaa₂” means reverse transcriptase) s (asused in a mutation, envelope gene e.g. sF134V) TFV tenofovir disoproxilfumarate YMDD Tyr Met Asp Asp-a motif in the polymerase protein; wherethe Met residue is designated residue number 204 of the reversetranscriptase

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation showing the partially doublestranded DNA HBV genome showing the overlapping open reading framesencoding surface (S), core (C), polymerase (P) and X gene.

FIG. 2 is a diagrammatic representation of the chemical structure ofADV.

FIG. 3 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient A during ADV treatment (sample A).

FIG. 4 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient A during ADV therapy (sample A).

FIG. 5 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Aduring ADV therapy (sample A).

FIG. 6 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient A during ADV treatment (sample B).

FIG. 7 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient A during ADV therapy (sample B).

FIG. 8 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Aduring ADV therapy (sample B).

FIG. 9 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient B during ADV and LMV treatment.

FIG. 10 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient B during ADV and LMV therapy.

FIG. 11 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Bduring ADV and LMV therapy.

FIG. 12 is a representation the HBV nucleotide sequence encoding thecatalytic region of the polymerase gene in samples from Patient C duringADV treatment.

FIG. 13 is a representation the deduced amino acid sequence of thecatalytic region of the polymerase gene in samples from Patient C duringADV therapy.

FIG. 14 is a representation the deduced amino acid sequence of theenvelope gene in samples from Patient C during ADV therapy.

FIG. 15 is a graphical representation of increase in HBV DNA levels(viral load) and ALT over time (days since the initiation of the firstantiviral therapy).

FIG. 16 is a representation the HBV nucleotide sequence encoding thecatalytic region of the polymerase gene in samples from Patient D duringADV and LMV treatment.

FIG. 17 is a representation the deduced amino acid sequence of thecatalytic region of the polymerase gene in samples from Patient D duringADV and LMV therapy.

FIG. 18 is a representation the deduced amino acid sequence of theenvelope gene in samples from Patient D during ADV and LMV therapy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the identification andisolation of nucleoside or nucleotide analog-resistant variants of HBVfollowing treatment of patients with either ADV or LMV or ETV or moreparticularly ADV and LMV or TFV and LMV, or ETV and optionally one ormore other nucleoside analogs or nucleotide analogs or other anti-HBVagents such as TFV or FTC. In particular, ADV or ADV and LMV or ETVtreated patients gave rise to variants of HBV exhibiting decreased orreduced sensitivity to ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV;LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV andFTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV andFTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV andTFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC andLMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV;ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof. With the proviso that the HBVvariant does not exhibit resistance to ETV alone or ETV or LMV alone.Reference herein to “decreased” or “reduced” in relation to sensitivityto ADV and/or LMV and/or FTC and/or TFV and/or ETV includes andencompasses a complete or substantial resistance to the nucleoside ornucleotide analog or other anti-HBV agents as well as partial resistanceand includes a replication rate or replication efficiency which is morethan a wild-type in the presence of a nucleoside or nucleotide analog orother anti-HBV agents. In one aspect, this is conveniently measured byan increase in viral load during treatment, or alternatively, there isno substantial decrease in HBV DNA viral load from pre-treatment HBV DNAlevels during treatment (i.e., non-response to treatment).

Accordingly, one aspect of the present invention provides an isolatedhepatitis B virus (HBV) variant wherein said variant comprises anucleotide mutation resulting in an amino acid addition, substitutionand/or deletion in a protein encoded by said variant and wherein saidvariant exhibits decreased sensitivity to one or more nucleoside ornucleotide analogs selected from the list consisting of ADV, LMV, TFV orFTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV;FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; and ADVand FTC and LMV and TFV and ETV.

In particular, the mutations are in the DNA polymerase or surfaceantigen (S gene product).

Accordingly, one aspect of the present invention contemplates anisolated Hepatitis B virus (HBV) variant wherein said variant comprisesa nucleotide mutation in a gene encoding a DNA polymerase resulting inat least one amino acid addition, substitution and/or deletion to saidDNA polymerase and wherein said variant exhibits decreased sensitivityto one or more nucleoside or nucleotide analogs selected from the listconsisting of ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV andTFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC;ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTCand LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV;ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMVand TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADVand FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof.

A further aspect of the present invention provides a method fordetermining the potential for an HBV to exhibit reduced sensitivity to anucleoside or nucleotide analog selected from ADV, LMV, TFV and FTC orETV and another analog except LMV or a combination thereof or optionallyother nucleoside or nucleotide analogs, said method comprising isolatingDNA or corresponding mRNA from said HBV and screening for a mutation inthe nucleotide sequence encoding HBV DNA polymerase resulting in atleast one amino acid substitution, deletion and/or addition in any oneor more of domains F and A through E or a region proximal thereto ofsaid DNA polymerase and associated with resistance or decreasessensitivity to one or more of ADV, LMV, TFV, FTC and/or wherein thepresence of such a mutation is an indication of the likelihood ofresistance to said one or more of ADV, LMV, TFV and/or FTC and/or ETVand another nucleoside or nucleotide analog except LMV.

Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated, the subject invention is notlimited to specific formulations of components, manufacturing methods,dosage regimens, or the like, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

It must be noted that, as used in the subject specification, thesingular forms “a”, “an” and “the” include plural aspects unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleoside or nucleotide analog” includes a single analog, as well astwo or more analogs; reference to “an HBV variant” includes reference totwo or more HBV variants; and so forth.

In describing and claiming the present invention, the followingterminology is used in accordance with the definitions set forth below.

The terms “analog”, “compound”, “active agent”, “pharmacologicallyactive agent”, “medicament”, “active” and “drug” are usedinterchangeably herein to refer to a chemical compound that induces adesired effect such as inhibit viral replication, infection,maintenance, assembly and/or the function of an enzyme such as HBV DNApolymerase. The terms also encompass pharmaceutically acceptable andpharmacologically active ingredients of those active agents specificallymentioned herein including but not limited to salts, esters, amides,prodrugs, active metabolites, analogs and the like. When the terms“analog”, “compound”, “active agent”, “pharmacologically active agent”,“medicament”, “active” and “drug” are used, then it is to be understoodthat this includes the active agent per se as well as pharmaceuticallyacceptable, pharmacologically active salts, esters, amides, prodrugs,metabolites, analogs, etc.

The present invention contemplates, therefore, compounds useful ininhibiting HBV replication, infection, maintenance, assembly and/or thefunction of an enzyme such as HBV DNA polymerase. Reference to an“analog”, “compound”, “active agent”, “pharmacologically active agent”,“medicament”, “active” and “drug” such as ADV, LMV, TFV, ETV or FTC; ADVand LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC andLMV; ETV and ADV; ETV and LMV; ETV and FTC; ETV and TFV; ADV and LMV andTFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV,ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV andFTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMVand TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; orADV and FTC and LMV and TFV and ETV and/or optionally other nucleosideor nucleotide analogs or other anti-HBV agents or combinations thereof.A “combination” also includes a two-part or more such as a multi-partanti-HBV therapeutic composition where the agents are providedseparately and given or dispensed separately or admixed together priorto dispensation.

The terms “effective amount” and “therapeutically effective amount” ofan agent as used herein mean a sufficient amount of the agent to providethe desired therapeutic or physiological effect of inhibiting HBVreplication, infection, maintenance, assembly and/or the function of anenzyme such as HBV DNA polymerase. Furthermore, an “effectiveHBV-inhibiting amount” or “effective symptom-ameloriating amount” of anagent is a sufficient amount of the agent to directly or indirectlyinhibit replication, infection, maintenance, assembly and/or thefunction of an enzyme such as HBV DNA polymerase. Undesirable effects,e.g. side effects, are sometimes manifested along with the desiredtherapeutic effect; hence, a practitioner balances the potentialbenefits against the potential risks in determining what is anappropriate “effective amount”. The exact amount required will vary fromsubject to subject, depending on the species, age and general conditionof the subject, mode of administration and the like. Thus, it may not bepossible to specify an exact “effective amount”. However, an appropriate“effective amount” in any individual case may be determined by one ofordinary skill in the art using only routine experimentation.

By “pharmaceutically acceptable” carrier, excipient or diluent is meanta pharmaceutical vehicle comprised of a material that is notbiologically or otherwise undesirable, i.e. the material may beadministered to a subject along with the selected active agent withoutcausing any or a substantial adverse reaction. Carriers may includeexcipients and other additives such as diluents, detergents, coloringagents, wetting or emulsifying agents, pH buffering agents,preservatives, and the like.

Similarly, a “pharmacologically acceptable” salt, ester, emide, prodrugor derivative of a compound as provided herein is a salt, ester, amide,prodrug or derivative that this not biologically or otherwiseundesirable.

The terms “treating” and “treatment” as used herein refer to reductionin severity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage in relationto HBV infection. Thus, for example, “treating” a patient involvesprevention of HBV infection as well as treatment of a clinically HBVsymptomatic individual by inhibiting HBV replication, infection,maintenance, assembly and/or the function of an enzyme such as HBV DNApolymerase. Thus, for example, the present method of “treating” apatient with HBV infection or with a propensity for one to developencompasses both prevention of HBV infection as well as treating HBVinfection or symptoms thereof. In any event, the present inventioncontemplates the treatment or prophylaxis of HBV infection.

“Patient” as used herein refers to an animal, preferably a mammal andmore preferably a primate including a lower primate and even morepreferably, a human who can benefit from the formulations and methods ofthe present invention. A patient regardless of whether a human ornon-human animal may be referred to as an individual, subject, animal,host or recipient. The compounds and methods of the present inventionhave applications in human medicine, veterinary medicine as well as ingeneral, domestic or wild animal husbandry. For convenience, an “animal”includes an avian species such as a poultry bird (including ducks,chicken, turkeys and geese), an aviary bird or game bird. The conditionin a non-human animal may not be a naturally occurring HBV infection butHBV-like infection may be induced.

As indicated above, the preferred animals are humans, non-human primatessuch as marmossets, baboons, orangatangs, lower primates such as tupia,livestock animals, laboratory test animals, companion animals or captivewild animals. A human is the most preferred target. However, non-humananimal models may be used.

Examples of laboratory test animals include mice, rats, rabbits, guineapigs and hamsters. Rabbits and rodent animals, such as rats and mice,provide a convenient test system or animal model as do primates andlower primates. Livestock animals include sheep, cows, pigs, goats,horses and donkeys. Non-mammalian animals such as avian species,zebrafish, amphibians (including cane toads) and Drosophila species suchas Drosophila melanogaster are also contemplated. Instead of a liveanimal model, a test system may also comprise a tissue culture system.

An “anti-HBV agent” includes a nucleoside or nucleotide analog, protein,chemical compound, RNA or DNA or RNAi or siRNA oligonucleotide (eitherDNA-derived or synthetic).

Preferably, the decreased sensitivity is in respect of ETV.Alternatively, the decreased sensitivity is in respect of ADV or LMV.Alternatively, the decreased sensitivity is in respect of TFV.Alternatively, the decreased sensitivity is in respect of FTC.Alternatively, the decreased sensitivity is in respect of ETV andanother nucleotide or nucleoside analog with the exception of LMV alone.Alternatively, the decreased sensitivity is in respect of ADV and TFVand optionally ETV. Alternatively, the decreased sensitivity is inrespect of LMV and TFV and optionally ETV. Alternatively, the decreasedsensitivity is in respect of ADV and FTC and optionally ETV.Alternatively, the decreased sensitivity is in respect to FTC and TFVand optionally ETV. Alternatively, the decreased sensitivity is inrespect of FTC and LMV and optionally ETV. Alternatively, the decreasedsensitivity is in respect of ADV and LMV and TFV and optionally ETV.Alternatively, the decreased sensitivity is in respect to ADV and TFVand FTC and optionally ETV. Alternatively, the decreased sensitivity isin respect to LMV and TFV and FTC and optionally ETV. Alternatively, thedecrease sensitivity is in respect of ADV and LMV and FTC and optionallyETV. Alternatively, the decreased sensitivity is in respect of ADV andFTC and TFV and LMV and optionally ETV. The present invention does notextend to an HBV variant exhibiting resistance or reduced sensitivity toETV alone or ETV and LMV alone.

Reference herein to “anti-HBV agents” includes nucleoside and nucleotideanalogs as well as immunological reagents (e.g. antibodies to HBVsurface components) and chemical, proteinaceous and nucleic acid agentswhich inhibit or otherwise interfere with viral replication,maintenance, infection, assembly or release. Reference herein to“nucleic acid” includes reference to a sense or antisense molecule, RNAor DNA, oligonucleotides and RNAi and siRNA molecules and complexescontaining same.

In addition to a mutation in the gene encoding DNA polymerase, due tothe overlapping nature of the HBV genome (FIG. 1), a correspondingmutation may also occur in the gene encoding the S gene encoding thesurface antigen (HBsAg) resulting in reduced interactivity ofimmunological reagents such as antibodies and immune cells to HBsAg. Thereduction in the interactivity of immunological reagents to a viralsurface component generally includes the absence of immunological memoryto recognize or substantially recognize the viral surface component. Thepresent invention extends, therefore, to an HBV variant exhibitingdecreased sensitivity to ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV;LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV andFTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV andFTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV andTFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC andLMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV;ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof or a reduced interactivity of animmunological reagent to HBsAg wherein the variant is selected forfollowing ADV and/or LMV combination or sequential treatment. The term“sequential” in this respect means ADV followed by LMV and/or TFV and/orETV, and/or FTC, LMV followed by ADV and/or TFV and/or FTC and/or ETV orETV followed by one or more of ADV, FTC, LMV and/or TFV, or multiplesequential administrations of each of ETV, ADV, LMV and/or TFV and/orFTC. With the proviso that the HBV variant does not exhibit resistanceto ETV alone or ETV or LMV alone.

A viral variant may, therefore, carry A mutation only in the DNApolymerase gene or both in the DNA polymerase gene and the S gene. Theterm “mutation” is to be read in its broadest context and includesmultiple mutations.

The present invention extends to a mutation and any domain of the HBVDNA polymerase and in particular regions F and G, and domains A throughto E provided said mutation leads to decreased sensitivity to ADV and/orLMV and/or TFV and/or ETV and/or FTC or combinations thereof.

In this specification, reference is particularly made to the conservedregions of the DNA polymerase as defined by domains A to E. Regions A toE are defined by the amino acid sequence set forth in Formula II inAustralian Patent No. 734831.

Preferably, the mutation results in an altered amino acid sequence inany one or more of domains F and G, and domains A through to E orregions proximal thereto of the HBV DNA polymerase.

Another aspect of the present invention provides an HBV variantcomprising a mutation in an overlapping open reading frame in its genomewherein said mutation is in a region defined by one or more of domains Fand G, and domains A through to E of HBV DNA polymerase and wherein saidvariant exhibits decreased sensitivity to ADV, LMV, TFV or FTC; ADV andLMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV;ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV andFTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV andTFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV andTFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV andFTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMVand TFV and ETV and/or optionally other nucleoside or nucleotide analogsor other anti-HBV agents or combinations thereof. With the proviso thatthe HBV variant does not exhibit resistance to ETV alone or ETV or LMValone.

Another preferred aspect of the present invention contemplates an HBVvariant comprising a mutation in the nucleotide sequence encoding HBsAgresulting in an amino acid addition, substitution and/or deletion insaid HBsAg wherein said variant exhibits decreased sensitivity to ADV,LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTCand TFV; FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMVand TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV andETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADVand FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV andLMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV;or ADV and FTC and LMV and TFV and ETV and/or optionally othernucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof. With the proviso that the HBV variant does notexhibit resistance to ETV alone or ETV or LMV alone.

More particularly, the present invention provides a variant HBVcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion or atruncation compared to a surface antigen from a reference or wild-typeHBV and wherein an antibody generated to the reference or wild-typesurface antigen exhibits reduced capacity for neutralizing said HBVvariant, said variant selected by exposure of a subject to ADV, LMV, TFVor FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV;FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADVand FTC and LMV and TFV and ETV and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combinations thereof.With the proviso that the HBV variant does not exhibit resistance to ETValone or ETV or LMV alone.

The term “combination therapy” means that both combinations of ADV, LMV,FTC, TFV, and/or ETV are co-administered in the same composition orsimultaneously in separate compositions. The term “sequential therapy”means that the two agents are administered within seconds, minutes,hours, days or weeks of each other and in either order. Sequentialtherapy also encompasses completing a therapeutic course with one orother of ADV, LMV, FTC, TFV or ETV and then completing a second or thirdor subsequent therapeutic courses with the other of ADV, LMV, FTC, TFVor ETV.

Accordingly, another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to LMV therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Yet another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to FTC therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to TFV therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Yet still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ETV therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Even yet another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV and LMV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Even still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV and TFV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Yet still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV and ETV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

A further aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to LMV and TFV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Yet a further aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to LMV and ETV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV and FTC therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Yet another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to TFV and FTC therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Still another aspect another aspect of the present inventioncontemplates an HBV variant comprising a surface antigen having an aminoacid sequence with a single or multiple amino acid substitution,addition and/or deletion or truncation compared to the pretreatment HBVand wherein the surface antigen of the variant HBV exhibits an alteredimmunological profile compared to the pretreatment HBV where the saidvariant HBV is selected for by exposure of a subject to TFV and ETVtherapy or therapy by one or more other nucleoside or nucleotide analogsor other anti-HBV agents.

Still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to FTC and LMV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Even another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to FTC and ETV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Even yet another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV and TFV and/or ETV therapy ortherapy by one or more other nucleoside or nucleotide analogs or otheranti-HBV agents.

Even still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV and TFV and/or ETV therapy ortherapy by one or more other nucleoside or nucleotide analogs or otheranti-HBV agents.

A further aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV and FTC and/or ETV therapy ortherapy by one or more other nucleoside or nucleotide analogs or otheranti-HBV agents.

Another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to FTC, LMV and TFV and/or ETV therapy ortherapy by one or more other nucleoside or nucleotide analogs or otheranti-HBV agents.

Yet another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, FTC and TFV and/or ETV therapy ortherapy by one or more other nucleoside or nucleotide analogs or otheranti-HBV agents.

Still yet another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV, FTC and TFV and/or ETV therapyor therapy by one or more other nucleoside or nucleotide analogs orother anti-HBV agents.

Preferably, the variants are in isolated form such that they haveundergone at least one purification step away from naturally occurringbody fluid. Alternatively, the variants may be maintained in isolatedbody fluid or may be in DNA form. The present invention alsocontemplates infectious molecular clones comprising the genome or partsthereof from a variant HBV. Furthermore, the present invention providesisolated components from the variant HBVs such as but not limited to anisolated HBsAg. Accordingly, the present invention provides an isolatedHBsAg or a recombinant form thereof or derivative or chemical equivalentthereof, said HBsAg being from a variant HBV selected by exposure of asubject to one or more of ADV, LMV, FTC, TFV and/or ETV or optionallyone or more nucleoside or nucleotide analogs or other anti-HBV agents.

More particularly, yet another aspect of the present invention isdirected to an isolated variant HBsAg or a recombinant or derivativeform thereof or a chemical equivalent thereof wherein said HBsAg or itsrecombinant or derivative form or its chemical equivalent exhibits analtered immunological profile compared to an HBsAg from a reference HBV,said HBsAg being from a variant HBV selected by exposure of a subject toone or more of ADV, LMV, FTC, TFV and/or ETV or optionally one or morenucleoside or nucleotide analogs or other anti-HBV agents.

Even more particularly, the present invention provides an isolatedvariant HBsAg or a recombinant or derivative form thereof or a chemicalequivalent thereof wherein said HBsAg or its recombinant or derivativeform or its chemical equivalent comprises an amino acid sequence with asingle or multiple amino acid substitution, addition and/or deletion ora truncation compared to an HBsAg from a reference HBV and wherein aneutralizing antibody directed to a reference HBV exhibits no or reducedneutralising activity to an HBV carrying said variant HBsAg, said HBsAgbeing from a variant HBV selected by exposure of a subject to one ormore of ADV, LMV, FTC, TFV and/or ETV or optionally one or morenucleoside or nucleotide analogs or other anti-HBV agents.

Preferred mutations in the HBV DNA polymerase include variants selectedfrom patients with HBV recurrence following ADV, LMV, TFV or FTC; ADVand LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC andLMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADVand FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETVand TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMVand TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADVand FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC andLMV and TFV and ETV and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combinations thereof. Nucleoside ornucleotide analogs or other anti-HBV agents may be indicated during,after or prior to a transplantation procedure (e.g. bone marrowtransplantation (BMT) or OLT) or following treatment of patientsdiagnosed with hepatitis. Following selection of variants, viral loadsare obtainable at levels similar to pre-treatment levels or increasewhile on therapy.

Useful mutants in the rt region include, in one embodiment, mutations atcodons rtN53, rtY54, rtL180, rtT181, rtT184, rtM204, rtN236, rtY124,rtH126, rtT128, rtS135, rtL180, rtS202, rtM204, rtH248, rtI53, rtS54,rtN122, rtM145, rtL180, rtM204, rtM250, rtN53, rtS85, rtS116, rtD134,rtN139, rtQ149, rtA181, rtS219, rtI233, rtN236 or rtM250 such as rtN53K,rtN53K/N, rtY54D, rtL180M, rtT181A/V rtT184S, rtM204V, and rtN236T, inanother embodiment include rtY124H, rtH126R, rtT128N, rtS135C, rtL180MrtS202C rtM204V, and rtH248N, or yet another embodiment include rtI53V,rtS54T, rtN122T, rtM145L, rtL180M, rtM204V and rtM250L, or still yetanother embodiment include rtN53D, rtS85A, rtS116P, rtD134V, rtN139E/K,rtQ149K, rtA181V, rtS219A, rtI233V, rtN236T, rtM250L or a combinationthereof or an equivalent mutation. Particularly useful mutants arertT184S, rtS202C, rtS219A, rtI233V, rtH248N or rtM250L

Such HBV variants are proposed to exhibit a decreased sensitivity toADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC andADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV;ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADVand LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMVand FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADVand ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC andLMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionallyother nucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combination thereof. It should benoted that the nomenclature system for amino acid positions is based onthe methionine residues in the YMDD motif being designated codon rtM204.This numbering system is different to that in Australian Patent No.734831 where the methionine residue in the YMDD motif within thepolymerase gene is designated codon 550. In this regard, rtL180M andrtM204V correspond to L526M and M550V, respectively, in AustralianPatent No. 734831. Corresponding mutations may also occur in envelopegenes such as in one or more of PreS1, PreS2 and S.

Another potential mode of action of ADV and other acyclic nucleosidephosphonates is that of immune stimulation (Calio et al., Antiviral Res.23: 77-89, 1994). A number of mutations resulted in changes in theenvelope gene detected in HBV variants which may be associated withimmune escape. These changes include in one embodiment include in oneembodiment include mutations at codons stT45, sL173, sL175, sI195,sT118, sP120, sP127, sI195, sT114, SI195, sS204, sI208, sS210, sV14,sG130, sM133, sW172, sS204 or sS210 such as sT45K, sL173L/F, sL175F,sI195M and in another embodiment include and sT118A, sP120T, sP127A andsI195M, or yet another embodiment include sT114P, sI195M, sS204N,sI208T, and sS210R, or still yet another embodiment include sV14A,sG130R, sM133T, sW172L, sS204G, sS210R or a combination thereof or anequivalent mutation, or a combination thereof or an equivalent mutation.

The identification of the variants of the present invention permits thegeneration of a range of assays to detect such variants. The detectionof such variants may be important in identifying resistant variants todetermine the appropriate form of chemotherapy and/or to monitorvaccination protocols, or develop new or modified vaccine preparations.

Still another aspect of the present invention contemplates a method fordetermining the potential for an HBV to exhibit reduced sensitivity toADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC andADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV;ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADVand LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMVand FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADVand ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC andLMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionallyother nucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents, said method comprising isolating DNAor corresponding mRNA from said HBV and screening for a mutation in thenucleotide sequence encoding HBV DNA polymerase resulting in at leastone amino acid substitution, deletion and/or addition in any one or moreof domains F and G, and A domains through to E or a region proximalthereto of said DNA polymerase and associated with resistance ordecreased sensitivity to ADV, LMV, TFV, or FTC; ADV and LMV; ADV andTFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV;ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV;TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV andLMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV andFTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETVand TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV andETV and/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof. With the proviso that the HBVvariant does not exhibit resistance to ETV alone or ETV or LMV alone.

Accordingly, another aspect of the present invention provides a methodfor determining the potential for an HBV to exhibit reduced sensitivityto a nucleoside or nucleotide analog selected from ADV, LMV, TFV and FTCand ETV and another nucleotide or nucleoside analog except LMV alone oroptionally other nucleoside or nucleotide analogs, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding HBV DNApolymerase resulting in at least one amino acid substitution, deletionand/or addition in any one or more of domains F and A through E or aregion proximal thereto of said DNA polymerase and associated withresistance or decreases sensitivity to one or more of ADV, LMV, TFV, FTCand/or ETV wherein the presence of such a mutation is an indication ofthe likelihood of resistance to said one or more of ADV, LMV, TFV and/orFTC and/or ETV and another analog except LMV alone. With the provisothat the HBV variant does not exhibit resistance to ETV alone or ETV orLMV alone.

Preferably, the assay detects one or more mutations at codons rtN53,rtY54, rtL180, rtT181, rtT184, rtM204, rtN236, rtY124, rtH126, rtT128,rtS135, rtL180, rtS202, rtM204, rtH248, rtI53, rtS54, rtN122, rtM145,rtL180, rtM204, rtM250, rtN53, rtS85, rtS116, rtD134, rtN139, rtQ149,rtA181, rtS219, rtI233, rtN236 or rtM250 such as rtN53K, rtN53K/N,rtY54D, rtL180M, rtT181A/V rtT184S, rtM204V, and rtN236T, in anotherembodiment include rtY124H, rtH126R, rtT128N, rtS135C, rtL180M rtS202CrtM204V, and rtH248N, or yet another embodiment include rtI53V, rtS54T,rtN122T, rtM145L, rtL180M, rtM204V and rtM250L, or still yet anotherembodiment include rtN53D, rtS85A, rtS116P, rtD134V, rtN139E/K, rtQ149K,rtA181V, rtS219A, rtI233V, rtN236T, rtM250L, or a combination thereof oran equivalent mutation is indicative of a variant wherein said variantexhibits a decreased sensitivity to ADV, LMV, TFV or FTC; ADV and LMV;ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV andADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC andTFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETVand LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETVand FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC andETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFVand ETV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combinations thereof and/or optionally othernucleoside or nucleotide analogs or other anti-HBV agents or combinationthereof.

Accordingly, another aspect of the present invention produces a methodfor determining whether an HBV strain exhibits reduced sensitivity to anucleoside or nucleotide analog or other anti-HBV agents, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding the DNApolymerase and/or a corresponding region of the S gene, wherein thepresence of a mutation selected from, in one embodiment includemutations at codons stT45, sL173, sL175, sI195, sT118, sP120, sP127,sI195, sT114, SI195, sS204, sI208, sS210, sV14, sG130, sM133, sW172,sS204 or sS210 such as sT45K, sL173L/F, sL175F, sI195M and in anotherembodiment include and sT118A, sP120T, sP127A and sI195M, or yet anotherembodiment include sT114P, sI195M, sS204N, sI208T, and sS210R, or stillyet another embodiment include sV14A, sG130R, sM133T, sW172L, sS204G,sS210R or a combination thereof or an equivalent mutation, in even stillanother embodiment, rtN53K, rtN53K/N, rtY54D, rtL180M, rtT181A/VrtT184S, rtM204V, and rtN236T, in another embodiment include rtY124H,rtH126R, rtT128N, rtS135C, rtL180M rtS202C rtM204V, and rtH248N, or yetanother embodiment include rtI53V, rtS54T, rtN122T, rtM145L, rtL180M,rtM204V and rtM250L, or still yet another embodiment include rtN53D,rtS85A, rtS116P, rtD134V, rtN139E/K, rtQ149K, rtA181V, rtS219A, rtI233V,rtN236T, rtM250L or a combination thereof or an equivalent mutation, orcombinations thereof or an equivalent one or more other mutation isindicative of a variant which exhibits a decreased sensitivity to ADV,LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTCand TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV and TFV; ADV and LMVand TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV andETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADVand FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV andLMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV;or ADV and FTC and LMV and TFV and ETV and/or optionally othernucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof.

A further aspect of the present invention produces a method fordetermining whether an HBV strain exhibits reduced sensitivity to anucleoside or nucleotide analog or other anti-HBV agents, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding the DNApolymerase and/or a corresponding region of the S gene, wherein thepresence of a mutation selected from, in one embodiment, mutations atcodons stT45, sL173, sL175, sI195, sT118, sP120, sP127, sI195, sT114,SI195, sS204, sI208, sS210, sV14, sG130, sM133, sW172, sS204 or sS210such as sT45K, sL173L/F, sL175F, sI195M and in another embodimentinclude and sT118A, sP120T, sP127A and sI195M, or yet another embodimentinclude sT114P, sI195M, sS204N, sI208T, and sS210R, or still yet anotherembodiment include sV14A, sG130R, sM133T, sW172L, sS204G, sS210R or acombination thereof or an equivalent mutation, in even still anotherembodiment, rtN53K, rtN53K/N, rtY54D, rtL180M, rtT181A/V rtT184S,rtM204V, and rtN236T, in another embodiment include rtY124H, rtH126R,rtT128N, rtS135C, rtL180M rtS202C rtM204V, and rtH248N, or yet anotherembodiment include rtI53V, rtS54T, rtN122T, rtM145L, rtL180M, rtM204Vand rtM250L, or still yet another embodiment include rtN53D, rtS85A,rtS116P, rtD134V, rtN139E/K, rtQ149K, rtA181V, rtS219A, rtI233V,rtN236T, rtM250L or a combination thereof or an equivalent mutation,combinations thereof or an equivalent one or more other mutation isindicative of a variant which exhibits a decreased sensitivity to ADV,LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTCand TFV; FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMVand TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV andETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADVand FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV andLMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV;or ADV and FTC and LMV and TFV and ETV and/or optionally othernucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof.

The detection of HBV or its components in cells, cell lysates, culturedsupernatant fluid and bodily fluid may be by any convenient meansincluding any nucleic acid-based detection means, for example, bynucleic acid hybridization techniques or via one or more polymerasechain reactions (PCRs). The term “bodily fluid” includes any fluidderived from the blood, lymph, tissue or organ systems including serum,whole blood, biopsy and biopsy fluid, organ explants and organsuspension such as liver suspensions. The invention further encompassesthe use of different assay formats of said nucleic acid-based detectionmeans, including restriction fragment length polymorphism (RFLP),amplified fragment length polymorphism (AFLP), single-strand chainpolymorphism (SSCP), amplification and mismatch detection (AMD),interspersed repetitive sequence polymerase chain reaction (IRS-PCR),inverse polymerase chain reaction (iPCR) and reverse transcriptionpolymerase chain reaction (RT-PCR), amongst others. Other forms ofdetection include Northern blots, Southern blots, PCR sequencing,antibody procedures such as ELISA, Western blot andimmunohistochemistry. A particularly useful assay includes the reagentsand components required for immobilized oligonucleotide- oroligopeptide-mediated detection systems.

One particularly useful nucleic acid detection system is the reversehybridization technique. In this technique, DNA from an HBV sample isamplified using a biotin or other ligand-labeled primer to generate alabeled amplificon. Oligonucleotides immobilized to a solid support suchas a nitrocellulose film are then used to capture amplified DNA byhybridization. Specific nucleic acid fragments are identified via biotinor the ligand. Generally, the labeled primer is specific for aparticular nucleotide variation to be detected. Amplification occursonly if the variation to be detected is present. There are many forms ofthe reverse hybridization assay and all are encompassed by the presentinvention.

Another aspect contemplated by the present invention provides a methodfor detecting a variant HBV exhibiting an altered immunological profilesaid method comprising isolating an HBV from a subject exposed to anucleoside or nucleotide analog selected from the listed consisting ofADV, LMV, TFV, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTCand ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV andFTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV andFTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV andTFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC andLMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV;ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof, and then contacting said HBVwith a panel of one or more antibodies to a surface antigen andscreening for any change in binding affinity or binding spectrum.

In a related embodiment, the present invention contemplates a method fordetecting a variant HBV exhibiting an altered immunological profile saidmethod comprising isolating a serum sample from a subject exposed to anucleoside or nucleotide analog selected from the listed consisting ofADV, LMV, TFV, ETV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTCand ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and LMV; ETV andFTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV andFTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV andTFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC andLMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV;ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof, and then contacting the serumwith a panel of HBV surface antigens or antibody-binding fragmentsthereof and screening for any change in binding affinity or bindingspectrum.

Detecting HBV replication in cell culture is particularly useful.

This and other aspects of the present invention is particularly amenableto microarray analysis such as to identify oligonucleotides includingsense and antisense molecules, RNAi or siRNA molecules or DNA orRNA-binding molecules which down-regulate genomic sequences ortranscripts of HBV. Microarray analysis may also be used to identifyparticular mutations in the HBV genome such as within the HBV DNApolymerase-coding region or the HBsAg-coding region.

Another aspect of the present invention contemplates a method fordetecting an agent which exhibits inhibitory activity to an HBV by:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from the HBV contained        in a plasmid vector and then transfecting said cells with said        construct;    -   contacting the cells, before, during and/or after transfection,        with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agents; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

In a preferred embodiment, the plasmid vector may include genes encodingpart or all of other viral vectors such as baculovirus or adenovirus(Ren and Nassal, 2001, supra) and the method comprises:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from the HBV contained        in or fused to an amount of a baculovirus genome or adenovirus        genome effective to infect cells and then infecting said cells        with said construct;    -   contacting the cells, before, during and/or after infection,        with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

In an alternative embodiment, the method comprises:

-   -   generating a continuous cell line comprising an infectious copy        of the genome of the HBV in a replication competent effective        amount such that said infectious HBV genome is stably integrated        into said continuous cell line such as but not limited to 2.2.15        or AD;    -   contacting the cells with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to the agent; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

The above-mentioned methods are particularly useful in identifying ordeveloping agents against HBV variants such as those carrying mutations,in one embodiment, mutations at codons rtN53, rtY54, rtL180, rtT181,rtT184, rtM204, rtN236, rtY124, rtH126, rtT128, rtS135, rtL180, rtS202,rtM204, rtH248, rtI53, rtS54, rtN122, rtM145, rtL180, rtM204, rtM250,rtN53, rtS85, rtS116, rtD134, rtN139, rtQ149, rtA181, rtS219, rtI233,rtN236 or rtM250 such as rtN53K, rtN53K/N, rtY54D, rtL180M, rtT181A/VrtT184S, rtM204V, and rtN236T, in another embodiment include rtY124H,rtH126R, rtT128N, rtS135C, rtL180M rtS202C rtM204V, and rtH248N, or yetanother embodiment include rtI53V, rtS54T, rtN122T, rtM145L, rtL180M,rtM204V and rtM250L, or still yet another embodiment include rtN53D,rtS85A, rtS116P, rtD134V, rtN139E/K, rtQ149K, rtA181V, rtS219A, rtI233V,rtN236T, rtM250L or a combination thereof or an equivalent mutation, ora combination thereof or an equivalent mutation; in a furtherembodiment, mutations at codons stT45, sL173, sL175, sI195, sT118,sP120, sP127, sI195, sT114, SI195, sS204, sI208, sS210, sV14, sG130,sM133, sW172, sS204 or sS210 such as sT45K, sL173L/F, sL175F, sI195M andin another embodiment include and sT118A, sP120T, sP127A and sI195M, oryet another embodiment include sT114P, sI195M, sS204N, sI208T, andsS210R, or still yet another embodiment include sV14A, sG130R, sM133T,sW172L, sS204G, sS210R or a combination thereof or an equivalentmutation.

Accordingly, another aspect of the present invention contemplates amethod for determining whether an HBV strain exhibits reducedsensitivity to a nucleoside or nucleotide analog or other potentialanti-HBV agent, said method comprising isolating DNA or correspondingmRNA from said HBV and screening for a mutation in the nucleotidesequence of the envelope genes or DNA polymerase gene selected from, inone embodiment, in one embodiment, mutations at codons rtN53, rtY54,rtL180, rtT181, rtT184, rtM204, rtN236, rtY124, rtH126, rtT128, rtS135,rtL180, rtS202, rtM204, rtH248, rtI53, rtS54, rtN122, rtM145, rtL180,rtM204, rtM250, rtN53, rtS85, rtS116, rtD134, rtN139, rtQ149, rtA181,rtS219, rtI233, rtN236 or rtM250 such as rtN53K, rtN53K/N, rtY54D,rtL180M, rtT181A/V rtT184S, rtM204V, and rtN236T, in another embodimentinclude rtY124H, rtH126R, rtT128N, rtS135C, rtL180M rtS202C rtM204V, andrtH248N, or yet another embodiment include rtI53V, rtS54T, rtN122T,rtM145L, rtL180M, rtM204V and rtM250L, or still yet another embodimentinclude rtN53D, rtS85A, rtS116P, rtD134V, rtN139E/K, rtQ149K, rtA181V,rtS219A, rtI233V, rtN236T, rtM250L, or a combination thereof or anequivalent mutation, or a combination thereof or an equivalent mutation;in a further embodiment, mutations at codons stT45, sL173, sL175, sI195,sT118, sP120, sP127, sI195, sT114, SI195, sS204, sI208, sS210, sV14,sG130, sM133, sW172, sS204 or sS210 such as sT45K, sL173L/F, sL175F,sI195M and in another embodiment include and sT118A, sP120T, sP127A andsI195M, or yet another embodiment include sT114P, sI195M, sS204N,sI208T, and sS210R, or still yet another embodiment include sV14A,sG130R, sM133T, sW172L, sS204G, sS210R or a combination thereof or anequivalent mutation and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combination thereof.

The detection of amino acid variants of DNA polymerase is convenientlyaccomplished by a range of amino acid detection techniques. Where an HBVvariant comprises an amino acid change, then such an isolate isconsidered a putative HBV variant having an altered DNA polymeraseactivity.

The present invention further contemplates agents which inhibit HBVvariants resistant to ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV;LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV andFTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV andFTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV andTFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC andLMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV;ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combinations thereof resistant HBV variants. Suchagents are particularly useful if long term treatment by ADV, LMV, FTC,TFV and/or ETV and/or optionally other nucleoside or nucleotide analogssuch as TFV is contemplated by the clinician. The agents may be DNA orRNA or proteinaceous or non-proteinaceous chemical molecules. Naturalproduct screening such as from plants, coral and microorganisms is alsocontemplated as a useful potential source of masking agents as is thescreening of combinatorial or chemical libraries. The agents may be inisolated form or in the form of a pharmaceutical composition orformulation and may be administered in place of or sequentially orsimultaneously with a nucleoside or nucleotide analog. Furthermore,rationale drug design is contemplated including solving the crystal orNMR structure of, for example, HBV DNA polymerase and designing agentswhich can bind to the enzyme's active site. This approach may also beadapted to other HBV components.

Accordingly, another aspect of the present invention contemplates amethod for detecting an agent which exhibits inhibitory activity to anHBV which exhibits resistance or decreased sensitivity ADV, LMV, TFV orFTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV;FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADVand FTC and LMV and TFV and ETV and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combinations thereofand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof, said method comprising:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from said HBV contained        in a plasmid vector and then transfecting said cells with said        construct;    -   contacting said cells, before, during and/or after transfection,        with the agent to be tested;    -   culturing said cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   subjecting the cells, cell lysates or culture supernatant fluid        to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of said agent.

Still another aspect of the present invention provides a method fordetecting an agent which exhibits inhibitory activity to an HBV whichexhibits resistance or decreased sensitivity to ADV, LMV, TFV or FTC;ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC andLMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADVand FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETVand TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMVand TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADVand FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC andLMV and TFV and ETV and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combinations thereof and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combination thereof, said method comprising:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from said HBV contained        in or fused to an amount of a baculovirus genome effective to        infect cells and then infecting said cells with said construct;    -   contacting said cells, before, during and/or after infection,        with the agent to be tested;    -   culturing said cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   subjecting the cells, cell lysates or culture supernatant fluid        to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of said agent.

Preferably, the HBV genome is stably integrated into the cells' genome.

Particularly useful cells are 2.2.15 cells (Price et al., Proc. Natl.Acad. Sci. USA 86(21): 8541-8544, 1989 or AD cells (also known asHepAD32 cells or HepAD79 cells [Ying et al., 2000 Spra].

Whilst the baculovirus vector is a particularly useful in the practiceof the present invention, the subject invention extends to a range ofother vectors such as but not limited to adenoviral vectors.

The present invention further extends to cell lines (e.g. 2.2.15 or ADcells) carrying genetic constructs comprising all or a portion of an HBVgenome or a gene or part of a gene therefrom.

The present invention also provides for the use of the subject HBVvariants to screen for anti-viral agents. These anti-viral agentsinhibit the virus. The term “inhibit” includes antagonizing or otherwisepreventing infection, replication, assembly and/or release or anyintermediate step. Preferred anti-viral agents include nucleoside ornucleotide analogs or anti-HBV agents, however, the present inventionextends to non-nucleoside molecules.

In addition, rational drug design is also contemplated to identify orgenerate chemical molecules which either mimic a nucleoside or whichinteract with a particular nucleotide sequence or a particularnucleotide. Combinatorial chemistry and two hybrid screening are some ofa number of techniques which can be employed to identify potentialtherapeutic or diagnostic agents.

In one example, the crystal structure or the NMR structure of polymeraseor the surface antigen is used to rationally design small chemicalmolecules likely to interact with key regions of the molecule requiredfor function and/or antigenicity. Such agents may be useful asinhibitors of polymerase activity and/or may alter an epitope on thesurface antigen.

Several models of the HBV polymerase have been prepared due to thesimilarity with reverse transcriptase from HIV (Das et al., J. Virol.75(10): 4771-4779, 2001; Bartholomeusz et al., Intervirology 40(5-6):337-342 1997; Allen et al., Hepatology 27(6): 1670-1677, 1998). Themodels of the HBV polymerase can be used for the rational drug design ofnew agents effective against HBV encoding the resistant mutations aswell as wild type virus. The rational drug that is designed may be basedon a modification of an existing antiviral agent such as the agent usedin the selection of the HBV encoding the mutations associated withresistance. Viruses or clones expressing HBV genomic material encodingthe mutations may also be used to screen for new antiviral agents.

In an alternative embodiment, the present invention also contemplates amethod for detecting an agent which exhibits inhibitory activity to anHBV polymerase in an in vitro polymerase assay. The HBV polymeraseactivity can be examined using established assays (Gaillard et al.,Antimicrob Agents Chemother. 46(4): 1005-1013, 2002; Xiong et al.,Hepatology 28(6): 1669-1673, 1998).

As indicated above, microarray technology is also a useful means ofidentifying agents which are capable of interacting with defined HBVinternal or external components. For example, arrays of HBV DNApolymerase or peptide fragments thereof carrying different amino acidvariants may be used to screen for agents which are capable of bindingor otherwise interacting with these molecules. This is a convenient wayof determining the differential binding patterns of agents between HBVvariants. Arrays of antibodies may also be used to screen for alteredHBsAg molecules. Microarrays are also useful in proteomic analysis toidentify molecules such as antibodies, interferons or cytokines whichhave an ability to interact with an HBV component. Microarrays of DNAand RNA molecules may also be employed to identify sense and antisensemolecules for genetic regions on the HBV genome or transcripts thereof.

The above methods are particularly useful in identifying an inhibitor ofan HBV resistant to or exhibiting reduced sensitivity to ADV, LMV, TFVor FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV;FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADVand FTC and LMV and TFV and ETV and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combinations thereofand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof. The present invention extends,therefore, to compositions of the inhibitors. The inhibitors may also bein the form of antibodies or genetic molecules such as ribozymes,antisense molecules and/or sense molecules for co-suppression or theinduction of RNAi or may be other nucleoside or nucleotide analogs orother anti-HBV agents or derivatives of known analogs. Reference to RNAiincludes reference to short, interfering RNAs (siRNA) and all RNAi-typemolecules may be DNA-derived or synthetic.

The term “composition” includes a “pharmaceutical composition” or aformulation.

The inhibitor is referred to below as an “active ingredient” or “activecompound” and may be selected from the list of inhibitors given above.

The composition may include an antigenic component of the HBV, adefective HBV variant or an agent identified through natural productscreening or rational drug design (including combinatorial chemistry).

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of encoding an aspartyl proteaseinhibitor. The vector may, for example, be a viral vector.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dilution medium comprising, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The proper fluidity can be maintained, for example, bythe use of superfactants. The preventions of the action ofmicroorganisms can be brought about by various anti-bacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thirmerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by filtered sterilization or other appropriate means ofsterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, suitable methods of preparation includevacuum drying and the freeze-drying technique which yield a powder ofactive ingredient plus any additionally desired ingredient.

When the active ingredient is suitably protected, it may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets. For oral therapeuticadministration, the active ingredient may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 200 mg of active compound. Alternativedosage amounts include from about 1 μg to about 1000 mg and from about10 μg to about 500 mg. These dosages may be per individual or per kgbody weight. Administration may be per hour, day, week, month or year.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter. A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavouring. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound(s) may be incorporated into sustained-release preparations andformulations.

As stated above, the present invention further extends to an isolatedHBsAg from the HBV variants herein described. More particularly, thepresent invention provides an HBsAg or a recombinant form thereof orderivative or chemical equivalent thereof. The isolated surfacecomponent and, more particularly, isolated surface antigen or itsrecombinant, derivative or chemical equivalents are useful in thedevelopment of biological compositions such as vaccine formulations.

Yet another aspect of the present invention provides a compositioncomprising a variant HBV resistant to ADV, LMV, TFV or FTC; ADV and LMV;ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV andADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC andTFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETVand LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETVand FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC andETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFVand ETV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combinations thereof and/or optionally othernucleoside or nucleotide analogs or other anti-HBV agents or an HBVsurface antigen from said variant HBV or a recombinant or derivativeform thereof or its chemical equivalent and one or more pharmaceuticallyacceptable carriers and/or diluents. Such a composition may be regardedas a therapeutic composition and is useful in generating an immuneresponse including a humoral response. Generally, the HBV variants are“defective” and in themselves are unable to cause a sustained infectionin a subject.

As indicated above, antibodies may be generated to the mutant HBV agentsand used for passive or direct vaccination against infection by theseviruses. The antibodies may be generated in humans or non-human animals.In the case of the latter, the non-human antibodies may need to bedeimmunized or more specifically humanized prior to use. Deimmunized mayinclude, for example, grafting complementarity determining regions(CDRs) from the variable region of a murine or non-human animal anti-HBVantibody onto a human consensus fragment antibody binding (Fab)polypeptide. Alternatively, amino acids defining epitopes in thevariable region of the antibody may be mutated so that the epitopes areno longer recognized by the human MHC II complex.

Insofar as ribozyme, antisense or co-suppression (RNAi) or siRNA orcomplexes thereof repression is concerned, this is conveniently aimed atpost-transcription gene silencing. DNA or RNA may be administered or acomplex comprising RNAi or a chemical analog thereof specific for HBVmRNA may be employed.

All such molecules may be incorporated into pharmaceutical compositions.

In another embodiment, the present invention provides a biologicalcomposition comprising a variant HBV or an HBsAg or L, M or S proteinsfrom said variant HBV or a recombinant or derivative form thereof or itschemical equivalent.

Generally, if an HBV is used, it is first attenuated. The biologicalcomposition according to this aspect of the present invention generallyfurther comprises one or more pharmaceutically acceptable carriersand/or diluents.

The biological composition may comprise HBsAg or like molecule from oneHBV variant or the composition may be a cocktail of HbsAgs or L, M or Sproteins or like molecules from a range of ADV- and/or LMV- and/or, FTC-and/or TFV-resistant HBV variants. Similar inclusions apply where thecomposition comprises an HBV.

The present invention is further directed to the use of defective HBVvariants in the manufacture of therapeutic vaccines to vaccinateindividuals against infection by HBV strains having a particularnucleotide sequence or encoding a particular polymerase or surfaceantigen or L, M or S proteins.

Examples of suitable vaccine candidates are defective forms of HBVvariants comprising a mutation selected from, in one embodiment, in oneembodiment, mutations at codons rtN53, rtY54, rtL180, rtT181, rtT184,rtM204, rtN236, rtY124, rtH126, rtT128, rtS135, rtL180, rtS202, rtM204,rtH248, rtI53, rtS54, rtN122, rtM145, rtL180, rtM204, rtM250, rtN53,rtS85, rtS116, rtD134, rtN139, rtQ149, rtA181, rtS219, rtI233, rtN236 orrtM250 such as rtN53K, rtN53K/N, rtY54D, rtL180M, rtT181A/V rtT184S,rtM204V, and rtN236T, in another embodiment include rtY124H, rtH126R,rtT128N, rtS135C, rtL180M rtS202C rtM204V, and rtH248N, or yet anotherembodiment include rtI53V, rtS54T, rtN122T, rtM145L, rtL180M, rtM204Vand rtM250L, or still yet another embodiment include rtN53D, rtS85A,rtS116P, rtD134V, rtN139E/K, rtQ149K, rtA181V, rtS219A, rtI233V,rtN236T, rtM250L or a combination thereof or an equivalent mutation, ora combination thereof or an equivalent mutation; in a furtherembodiment, mutations at codons stT45, sL173, sL175, sI195, sT118,sP120, sP127, sI195, sT114, SI195, sS204, sI208, sS210, sV14, sG130,sM133, sW172, sS204 or sS210 such as sT45K, sL173L/F, sL175F, sI195M andin another embodiment include and sT118A, sP120T, sP127A and sI195M, oryet another embodiment include sT114P, sI195M, sS204N, sI208T, andsS210R, or still yet another embodiment include sV14A, sG130R, sM133T,sW172L, sS204G, sS210R or a combination thereof or an equivalentmutation.

In one embodiment, for example, an HBV variant may be identified havinga particular mutation in its polymerase conferring resistance ordecreased sensitivity to a nucleoside or nucleotide analog. This variantmay then be mutated to render it defective, i.e. attenuated or unable tocause infection. Such a defective, nucleoside or nucleotideanalog-resistant virus may then be used as a therapeutic vaccine againstvirulent viruses having the same mutation in its polymerase.

The subject invention extends to kits for assays for variant HBVresistant to ADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV andTFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC;ETV and TFV; ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTCand LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV;ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMVand TFV; ADV and ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADVand FTC and LMV and ETV; or ADV and FTC and LMV and TFV and ETV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof. Such kits may, for example, contain thereagents from PCR or other nucleic acid hybridization technology orreagents for immunologically based detection techniques. A particularlyuseful assay includes the reagents and components required forimmobilized oligonucleotide- or oligopeptide-mediated detection systems.

Still another aspect of the present invention contemplates a method fordetermining the potential for an HBV to exhibit reduced sensitivity toADV, LMV, TFV or FTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC andADV; FTC and TFV; FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV;ADV and LMV and TFV; or ADV and FTC and TFV; TFV and FTC and LMV; ADVand LMV and ETV, ADV and ETV and TFV; ETV and LMV and TFV; ADV and LMVand FTC; ADV and FTC and LMV and TFV; ETV and FTC and LMV and TFV; ADVand ETV and LMV and TFV; ADV and FTC and ETV and TFV; ADV and FTC andLMV and ETV; or ADV and FTC and LMV and TFV and ETV and/or optionallyother nucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combination thereof, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding HBV DNApolymerase resulting in at least one amino acid substitution, deletionand/or addition in any one or more of domains F and G, and domains Athrough to E or a region proximal thereto of said DNA polymerase andassociated with resistance or decreased sensitivity to ADV, LMV, TFV orFTC; ADV and LMV; ADV and TFV; LMV and TFV; FTC and ADV; FTC and TFV;FTC and LMV; ETV and ADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV;or ADV and FTC and TFV; TFV and FTC and LMV; ADV and LMV and ETV, ADVand ETV and TFV; ETV and LMV and TFV; ADV and LMV and FTC; ADV and FTCand LMV and TFV; ETV and FTC and LMV and TFV; ADV and ETV and LMV andTFV; ADV and FTC and ETV and TFV; ADV and FTC and LMV and ETV; or ADVand FTC and LMV and TFV and ETV and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combinations thereof,wherein the presence of such a mutation is an indication of thelikelihood of resistance to said ADV, LMV, TFV or FTC; ADV and LMV; ADVand TFV; LMV and TFV; FTC and ADV; FTC and TFV; FTC and LMV; ETV andADV; ETV and FTC; ETV and TFV; ADV and LMV and TFV; or ADV and FTC andTFV; TFV and FTC and LMV; ADV and LMV and ETV, ADV and ETV and TFV; ETVand LMV and TFV; ADV and LMV and FTC; ADV and FTC and LMV and TFV; ETVand FTC and LMV and TFV; ADV and ETV and LMV and TFV; ADV and FTC andETV and TFV; ADV and FTC and LMV and ETV; or ADV and FTC and LMV and TFVand ETV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combinations thereof. With the proviso that theHBV variant does not exhibit resistance to ETV alone or ETV or LMValone.

An assessment of a potential viral variant is important for selection ofan appropriate therapeutic protocol. Such an assessment is suitablyfacilitated with the assistance of a computer programmed with software,which inter alia adds input codes for at least two features associatedwith the viral variants to provide a value corresponding to theresistance or sensitivity of a viral variant to a particular chemicalcompound or immunological agent. The value can be selected from (a) theability to exhibit resistance for reduced sensitivity to a particularcompound or immunological agent; (b) an altered DNA polymerase fromwild-type HBV; (c) an altered surface antigen from wild-type HBV; or (d)morbidity or recovery potential of a patient. Thus, in accordance withthe present invention, the values for such features are stored in amachine-readable storage medium, which is capable of processing the datato provide a value for a particular viral variant or a biologicalspecimen comprising same.

Thus, in another aspect, the invention contemplates a computer programproduct for assessing the likely usefulness of a viral variant orbiological sample comprising same for determining an appropriatetherapeutic protocol in a subject (FIG. 15), said product comprising:

-   -   (1) code that receives as input code for at least two features        associated with said viral agents or biological sample        comprising same, wherein said features are selected from:        -   (a) the ability to exhibit resistance for reduced            sensitivity to a particular compound or immunological agent;        -   (b) an altered DNA polymerase from wild-type HBV;        -   (c) an altered surface antigen from wild-type HBV; or        -   (d) morbidity or recovery potential of a patient;    -   (2) code that adds said input code to provide a sum        corresponding to a value for said viral variants or biological        samples; and    -   (3) a computer readable medium that stores the codes.

In a related aspect, the invention extends to a computer for assessingthe likely usefulness of a viral variant or biological sample comprisingsame in a subject, wherein said computer comprises:

-   -   (1) a machine-readable data storage medium comprising a data        storage material encoded with machine-readable data, wherein        said machine-readable data comprise input codes for at least two        features associated with said viral variant or biological        sample; wherein said features are selected from:—        -   (a) the ability to exhibit resistance for reduced            sensitivity to a particular compound or immunological agent;        -   (b) an altered DNA polymerase from wild-type HBV;        -   (c) an altered surface antigen from wild-type HBV; or        -   (d) morbidity or recovery potential of a patient;    -   (2) a working memory for storing instructions for processing        said machine-readable data;    -   (3) a central-processing unit coupled to said working memory and        to said machine-readable data storage medium, for processing        said machine readable data to provide a sum of said input code        corresponding to a value for said compound(s); and    -   (4) an output hardware coupled to said central processing unit,        for receiving said value.

Any general or special purpose computer system is contemplated by thepresent invention and includes a processor in electrical communicationwith both a memory and at least one input/output device, such as aterminal. FIG. 15 shows a generally suitable computer system. Such asystem may include, but is not limited, to personal computers,workstations or mainframes. The processor may be a general purposeprocessor or microprocessor or a specialized processor executingprograms located in RAM memory. The programs may be placed in RAM from astorage device, such as a disk or pre-programmed ROM memory. The RAMmemory in one embodiment is used both for data storage and programexecution. The computer system also embraces systems where the processorand memory reside in different physical entities but which are inelectrical communication by means of a network.

In an alternative embodiment, the program screens for a mutationselected from, in one embodiment, in one embodiment, mutations at codonsrtN53, rtY54, rtL180, rtT181, rtT184, rtM204, rtN236, rtY124, rtH126,rtT128, rtS135, rtL180, rtS202, rtM204, rtH248, rtI153, rtS54, rtN122,rtM145, rtL180, rtM204, rtM250, rtN53, rtS85, rtS116, rtD134, rtN139,rtQ149, rtA181, rtS219, rtI233, rtN236 or rtM250 such as rtN53K,rtN53K/N, rtY54D, rtL180M, rtT181A/V rtT184S, rtM204V, and rtN236T, inanother embodiment include rtY124H, rtH126R, rtT128N, rtS135C, rtL180MrtS202C rtM204V, and rtH248N, or yet another embodiment include rtI53V,rtS54T, rtN122T, rtM145L, rtL180M, rtM204V and rtM250L, or still yetanother embodiment include rtN53D, rtS85A, rtS116P, rtD134V, rtN139E/K,rtQ149K, rtA181V, rtS219A, rtI233V, rtN236T, rtM250L or a combinationthereof or an equivalent mutation or a combination thereof or anequivalent mutation; in a further embodiment, mutations at codons stT45,sL173, sL175, sI195, sT118, sP120, sP127, sI195, sT114, 51195, sS204,sI208, sS210, sV14, sG130, sM133, sW172, sS204 or sS210 such as sT45K,sL173L/F, sL175F, sI195M and in another embodiment include and sT118A,sP120T, sP127A and sI195M, or yet another embodiment include sT114P,sI195M, sS204N, sI208T, and sS210R, or still yet another embodimentinclude sV14A, sG130R, sM133T, sW172L, sS204G, sS210R or a combinationthereof or an equivalent mutation.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Overlapping Genome of HBV

The overlapping genome of HBV is represented in FIG. 1. The geneencoding DNA polymerase (P), overlaps the viral envelope genes, Pre-S1and Pre-S2, and partially overlaps the X and core (C) genes. The HBVenvelope comprises small, middle and large proteins HBV surfaceantigens. The large protein component is referred to as the HBV surfaceantigen (HBsAg) and is encoded by the S gene sequence. The Pre-S1 andPre-S2 gene sequences encode the other envelope components.

EXAMPLE 2 Patients on ADV Treatment and Analysis of HBV DNA

Patient A: During ADV treatment, unique HBV mutations were detected byPCR amplification sequencing using the methods described in Example 4;(Table 4). There was an evolution in the quasspecies detected. Themutation profile initially selected on ADV (sample A) includes theunique mutations at rtN53K, rtY54D, rtL180M, rtT184S, and rtM204V. A keyunique mutation is rtT1845. Subsequently, a different ADV resistanceprofile was detected in sample B rtN53K/N, rtL180M, rtT181A/V, andrtN236T. Key unique mutations are rtT181A/V, and rtN236T detectedpreviously in ADV resistant patients (Table 4, FIGS. 3, 4, 5 6, 7 and8).

The changes in the HBsAg include sT45K, sL175F, sI195M in sample A andin the subsequent sample B sT45K and sL173L/F were detected. Theseunique changes were compared to reference sequences from each of theeight genotypes A-H as well as a consensus sequence from pretreatmentsamples to determine unique changes

Patient B: The HBV mutations during ADV treatment are listed in Table 5and FIGS. 9, 10, and 11. Key unique changes in the rt region of the HBVDNA polymerase include rtS202C and rtH248N. Other changes in the HBVpolymerase while on ADV treatment include rtY124H, rtH126R, rtT128N,rtS135C, rtL180M and rtM204V. The changes in the HBsAg while on ADVtreatment include sT118A, sP120T, sP127A and sI195M.

Patient C: The HBV mutations during ADV treatment are listed in Table 6and FIGS. 12, 13, and 14. The key unique change in the rt region of theHBV DNA polymerase included rtM250L. Other unique changes in the HBVpolymerase while on ADV treatment include rtI53V, rtS54T, rtN122T,rtM145L, rtL180M and rtM204V. The changes in the HBsAg while on ADVtreatment include sT114P, sI195M, sS204N, sI208T, and sS210R.

Patient D: This patient had initially selected ADV resistant mutationsat rtN236T (Angus et al., Gastroenterology. 125(2):292-7. 2003). Thispatient was subsequently treated with ADV and LMV. There was avirological breakthrough during the combination antiviral therapy (referto FIG. 15 for a graphical representation of increase in HBV DNA levels(viral load) and ALT over time (days since the initiation of the firstantiviral therapy). The HBV mutations during dual ADV and LMV treatmentare listed in Table 7 and FIGS. 16, 17 and 18. By PCR amplification ofthe HBV genome and direct sequencing the unique changes in the rt regionof the HBV DNA polymerase include, rtD134V, rtN139E/K, rtI233V, rtM250L.These occurred in association with the known ADV resistant mutations atrtA181V and rtN236T. The key unique mutations include rtI233V andrtM250L. All unique changes in the HBV polymerase while on ADV And LMVtreatment include rtN53D, rtS116P, rtD134V, rtN139E, rtA181V, rtS219A,rtI233V, rtN236T, rtM250L. Clonal analysis was performed on a PCRamplified product encoding the polymerase genes, from HBV isolated atthe time of virological breakthrough (see Example 15). A further keymutations were detected in this PCR amplified product at rtS85A othermutations at rtN139K/E and rtQ149K were also detected as minor species(Table 7 footnote).

The changes in the HBsAg while on ADV and LMV treatment include sV14A,sG130R, sM133T, sW172L, sS204G, sS210R.

EXAMPLE 3 Detection of Viral Markers

Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg),anti-HBe and hepatitis B core antigen (HBcAg) specific IgG and IgM weremeasured using commercially available immunoassays (Abbott Laboratories,North Chicago, Ill., USA). Hepatitis B viral DNA levels were measuredusing a capture hybridization assay according to the manufacturer'sdirections (Digene Hybrid Capture II, Digene Diagnostics Inc.,Beltsville, Md.). The manufacturers stated cut-off for detecting HBVviremia in clinical specimens was 0.7×10⁶ copies/ml or 2.5 pg/ml[Hendricks et al., Am J Clin Pathol 104: 537-46, 1995]. HBV DNA levelscan also be quantitated using other commercial kits such as Cobasamplification HBV monitor kit (Roche).

EXAMPLE 4 Sequencing of HBV DNA

HBV DNA was extracted from 100 μl of serum as described previously byAye et al., J. Hepatol. 26: 1148-1153, 1997. Oligonucleotides weresynthesized by Geneworks, Adelaide, Australia. Amplification of the HBVpolymerase gene or the entire HBV genome has been described by Aye etal., 1997, supra and Ayres et al., Methods Mol Med. 95:125-49, 2004.

The specific amplified products were purified using PCR purificationcolumns such as from MO BIO Laboratories Inc (La Jolla, Calif.) anddirectly sequenced using Big Dye terminator Cycle sequencing ReadyReaction Kit (Perkin Elmer, Cetus Norwalk, Conn.). The PCR primers wereused as sequencing primers, OS1 5′-GCC TCA TTT TGT GGG TCA CCA TA-3′ (nt1408-1430) [SEQ ID NO:1], TTA3 5′-AAA TTC GCA GTC CCCAAA-3′(nt2128-2145) [SEQ ID NO:2], JM 5′-TTG GGG TGG AGC CCT CAGGCT-3′(nt1676-1696) [SEQ ID NO:3], TTA4 5′-GAA AAT TGG TAA CAG CGG-3′(nt 2615-2632) [SEQ ID NO:4], OS2 5′ TCT CTG ACA TAC TTT CCA AT 3′ (nt2798-2817) [SEQ ID NO:5], to sequence the internal regions of the PCRproducts.

EXAMPLE 5 Adefovir Dipivoxil (ADV)

ADV (formerly Bis-pom PMEA)) is a potent inhibitor of HBV replication.The structure of ADV is shown in FIG. 2 and its synthesis is describedby Benzaria et al., J Med Chem. 39: 4958-4965, 1996).

EXAMPLE 6 In Vitro Analysis of Antiviral Resistance

The sensitivity/resistance profile of HBV mutants to adefovir, tenofovirand other antiviral agents was examined in vitro using recombinantHBV/baculovirus. The procedure for analysing the resistance profile isoutlined in the following Examples 7-14.

EXAMPLE 7 Cell Culture

Sf21 insect cells were maintained in supplemented Grace's insect mediumfurther supplemented with 10% v/v heat-inactivated fetal bovine serum(Gibco BRL, Gaithersburg, Md.) in humidified incubator at 28 C with CO₂.HepG2 cells were maintained in minimal essential medium supplementedwith 10% v/v heat-inactivated fetal bovine serum (MEM-FBS). HepG2 cellswere grown in humidified 37° C. incubators at 5% v/v CO₂.

EXAMPLE 8 Preparation of HBV/Baculovirus Transfer Vector with SpecificPoint Mutations

The recombinant HBV/baculovirus system used for antiviral testing hasbeen previously described (Delaney et al., Antimicrob Agents Chemother45(6): 1705-1013, 2001). In brief, the recombinant transfer vector wascreated by excising a fragment containing the 1.3×HBV genome constructand cloning it into the multiple cloning region of a baculovirus vectorpBlueBac4.5 (Invitrogen, Carlsbad, Calif.). Point mutations were createdby site directed mutagenesis using the commercial kits according to themanufacturers specifications (QuikChange, Stratagene). The nucleotidesequence of the plasmid and the point mutations generated by sitedirected mutagenesis were confirmed by sequencing using the ABI PrismBig Dye Terminator Cycle Sequencing Ready Reaction Kit according to themanufacturer's specifications (Perkin Elmer, Cetus Norwalk, Conn.). Thepanel of HBV mutants was generated from a wild type (WT) genotype D HBVisolate by site-directed mutagenesis. Mutants encoded either WT HBVpolymerase or polymerase with the amino acid substitutions listed inTable 8 in an HBeAg negative (precore [PC] 01896A mutant) background.Other HBV mutants can be made and tested for antiviral sensitivity in asimilar manner.

EXAMPLE 9 Generation of Recombinant Baculoviruses Containing the 1.3 HBVConstruct

Purified recombinant transfer vector and linear AcMNPV baculovirus DNAwere co-transfected into Sf21 cells using the BacNBlue transfection kitfrom Invitrogen (Carlsbad, Calif.); recombinant viruses were isolated byplaque assay according to the manufacturer's instructions. A series ofrecombinant viruses were amplified from isolated plaques by infecting100-mm dishes of Sf21 cells. Viral DNA was extracted from amplifiedviruses using standard procedures. Purified viral DNA was digested withrestriction enzymes and then fractionated by electrophoresis in a 1% v/vagarose gel. Southern blotting was performed to determine which virusisolates contained the intact 1.3 HBV construct. A Boehringer MannheimRandom Prime DNA Labeling kit (Indianapolis, Ind.) was used to generate[P³²]-radiolabeled probes. A full-length double-stranded HBV genome wasused as a template for all radiolabeled probes. Viral DNA sequence wasconfirmed by PCR amplification and sequencing of the polymerasecatalytic region.

EXAMPLE 10 Preparative Baculovirus Amplification and Purification

Baculoviruses were amplified by infecting suspension cultures of Sf21cells in log phase at a multiplicity of infection (moi) of 0.5 pfu/cell.Infections were allowed to proceed until a majority of the cells in theflasks showed visible signs of infection (four to five days). Virionswere concentrated from infected Sf21 medium by centrifugation at80,000×g and purified through a 20-60% w/v sucrose gradient. Purifiedvirus was titrated in quadruplicate in Sf21 cells by end-point dilution.An aliquot of each high titer stock was used for DNA extraction. Thepolymerase gene was amplified and sequenced to confirm the presence ofthe site-directed mutagenesis.

EXAMPLE 11 Infection (Ie., Transduction) of HepG2 Cells with RecombinantHBV Expressing Baculovirus with Antiviral Agents

HepG2 cells were seeded at approximately 20-40% confluency and then weregrown for 16-24 hours before infection. On the day of infection,triplicate plates of cells were trypsinized, and viable cell number wasdetermined with a hemocytometer using Trypan blue exclusion. Averagecell counts were calculated and used to determine the volume ofhigh-titer viral stock necessary to infect cells at the indicated moi.HepG2 cells were washed one time with serum-free MEM to remove traces ofserum. Baculovirus was diluted into MEM without serum to achieve theappropriate moi using volumes of 1.0, 0.5, and 0.25 ml to infect 100-mm,60 mm, and 35-mm dishes, respectively. Baculovirus was adsorbed to HepG2cells for one hour at 37° C. with gentle rocking every 15 minutes toensure that the inoculum was evenly distributed. The inoculum was thenaspirated and HepG2 cells were washed two times with phosphate-bufferedsaline and refed MEM-FBS with or without various concentrations ofagents.

Replicate HepG2 cell cultures were transduced with WT or mutant HBV bymeans of baculovirus vectors, and then exposed continuously to differentconcentrations of each drug 7 days. For each assay, five differentconcentrations of each drug were tested in duplicate. Drugconcentrations for nucleoside analogs (LMV, FTC, L-dT and ETV) were (innM) 1, 10, 100, 1,000 and 10,000. For the nucleotide analogs (ADV andTFV) concentrations used were 100, 500, 1,000, 5000 and 10,000 nM.Duplicate untreated cultures served as controls. After 7 days culture,DNA was extracted and analysed.

EXAMPLE 12 Analysis of Secreted HBV Antigen

Detection of hepatitis Be antigen (HBeAg) was performed byradioimmunoassay and microparticle enzyme immunoassay using kitspurchased from Abbott Laboratories (Abbott Park, Ill., USA). Medium fromHepG2 cells was collected, centrifuged at 6,000 g to remove cellulardebris, transferred to clean tubes, and stored at 20° C. until analysis.HBeAg values are expressed as fold of positive control. Medium sampleswere diluted appropriately so that radioimmunoassay results were belowpositive control values for HBeAg.

EXAMPLE 13 Detection of Intracellular Replicative Intermediates

HBV core particles were isolated from the cytoplasmic fraction of HepG2cells lysed in 0.5% w/v NP-40. Cytoplasmic extracts were adjusted to 10mmol/1 McCl2 and unprotected DNA was removed by an incubation to 500g/ml Proteinase K for 1.5 hours at 37° C. HBV DNA in the samples wasthen extracted using commercial DNA extraction kits such as Qiagen (DNAextraction) or in-house methods using sequential phenol and chloroformextractions, and the nucleic acids were recovered by ethanolprecipitation. Nucleic acids were resuspended in 50 μl/1 TE (10 mmol/1Tris, 1 mmol/l ethylenediaminetetraacetic acid), normalized by OD260,and digested with 100 g/ml RNase (Boehringer Mannheim, Indianapolis,Ind.) for one hour at 37° C. before analysis by real-time PCR orelectrophoresis and Southern blotting using a full length [³²P]-labelledHBV and autoradiographed with the aid of Bio-Rad phosphorimagingscreens. Images were recorded and analysed using a Bio-Rad Model FXMolecular Imager equipped with “Quantity One” software.

The amount of HBV DNA in drug-treated cells was expressed as a fractionof the mean amount of HBV DNA in untreated controls, defined as 1.0based on image densities. Pairs of values from duplicates were averagedto give a single value for each of three independent assays, from whichmeans and standard deviations were calculated. The resulting data wereanalysed using TableCurve2D, which generated dose-response plots fromwhich EC50, EC90 and AUC (area under curve) were estimated.

EXAMPLE 14 Antiviral Testing Performed with Wild-Type andHBV/Baculovirus Encoding Polymerase Mutations

The effect of the antiviral agents on the panel of mutants is shown inTable 9, Antiviral testing shows that the mutations previously selectedon ETV at codons 184 and 202 in association with the LMV resistantmutants also affect sensitivity to ADV and TDF. The 184 and 202mutations were selected by patients on ADV treatment alone (see patientsA, and B). HBV encoding other mutations will be tested using similarmethodology.

EXAMPLE 15 Clonal Analysis of HBV Polymerase Mutants Selected DuringAntiviral Therapy

Clonal analysis was performed at the time of the virologicalbreakthrough on LMV and ADV therapy to determine if multiple HBVmutations are on the same genome for patient D. The catalytic domains ofthe reverse transcriptase/polymerase were amplified separately usingprimers OS1 and OS2 (refer to Example 4) sequenced as previouslydescribed Ayres et al., 2004 in supra). The OS1/OS2 PCR productsobtained from these samples were cloned into PCRScript Amp SK+(Stratagene) as per the manufacturers instructions. PCR amplificationwas carried out directly from each clone and the PCR product wassequenced for each sample as above, using the OS1/OS2 PCR primers assequencing primers. During failure of ADV monotherapy, the patientpreviously selected rtN236T and rtA181 T. At the time of dual ADV andLMV resistance the mutation profile detected by amplification of theentire genome and direct sequencing included rtN53D, rtS116P, rtD134V,rtN139E, rtA181V, rtI233V, rtN236T, rtM250L. The rtN236T and the rtA181Twere detected in 100% of clones. Two major clonal profiles weredetected: rtN139E+rtQ149K+rtM250L+rtI233V in 55% of clones andrtS85A+rtN139K+rtN238D in 45% of the clones, respectively. The keymutation at rtS85A and the mutations at rtN139E/K and rtQ149K were notdetected by direct sequencing in the initial amplification of the HBVgenome and PCR sequencing but was detected by directed sequencing of theclones and also the PCR product used for clonal analysis.

The rtM250L mutation could affect sensitivity to entecavir as previouslyanother mutation at codon rt250 was selected during ETV treatment. ThertI233V is located in a similar position in a sequence alignment withthe HIV reverse transcriptase to the HIV codon 215 (Batholomeusz et al.,Antivir Ther. 9(4149-6, 2004) The rtM204I/V+/−rtL180M LMV resistancemutations were not detected in the clonal analysis at the time ofvirological breakthrough on combination ADV and LMV therapy.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 4 Patient A HBV Polymerase and envelope mutations detected duringADV therapy Treatment HBV RT Mutations HBsAg Mutations LMV rtM132LsT189I/T Pre ADV rtL/M180M sL/P192L rtA/V200A sT195T/I M/I204M sW/L196LADV rtN53K sT45K rtY54D sL175F rtL180M sI195M rtT184T/S rtA200A/VrtM204M/I/V ADV rtN53K sT45K (Sample A) rtY54D sL175F rtL180M sI195MrtT184S rtM204V ADV rtN53K/N sT45K (Sample B) rtL180M sL173L/F rtT181A/VrtN236T

TABLE 5 Patient B RT and Polymerase mutations detected during ADVtherapy Treatment HBV RT Mutations HBsAg Mutations ADV rtY124H sT118ArtH126R, sP120T rtT128N sP127A rtS135C, sI195M rtL180M rtS202C, rtM204VrtH248N

TABLE 6 Patient C RT and Polymerase mutations detected during ADVtherapy Treatment HBV RT Mutations HBsAg Mutations ADV rtI16T sT114PrtI53V sI195M rtS54T sS204N rtN122T sI208T rtM145L sS210R rtL180MrtM204V rtM250L~

TABLE 7 Patient D RT and Polymerase mutations detected by direct PCRamplification sequencing during ADV and LMV therapy* Treatment HBV RTMutations HBsAg Mutations ADV and LMV rtN53D, sL173F rtS116P, sS204G,rtD134V, sS210R rtL180M, rtA181V, rtN238D ADV AND LMV* rtN53D, sV14A,(1946 days since start rtS116P, sG130R, of initial therapy) rtD134V,sM133T, rtN139E, sW172L, rtA181V sS204G, rtS219A, sS210R rtI233V,rtN236T, rtM250L *In the clonal analysis the key mutation at rtS85A, andother mutations at rtN139K/E and rtQ149K were also detected as minorspecies.

TABLE 8 Description of HBV Mutants Code for HBV Reverse transcriptase(rt) amino acid residue number mutants 169 173 180 184 202 204 250 WT IV L T S M M L180M double I V M T S V M L180M quad I V M G I V M V173Ltriple I L M T S V M

TABLE 9 Antiviral Results Summary Poly- merase Change LMV FTC ADV TFVETV L-dT none EC50 1.0 1.0 1.0 1.0 1.0 1.0 (WT) EC90 1.0 1.0 1.0 1.0 1.01.0 AUC 1.0 1.0 1.0 1.0 1.0 1.0 L180M EC50 >4,000 >1,370 2.9 2.3 70.04.7 double EC90 >435 >263 1.5 2.6 >12,048 >41 AUC 353.1 761.4 1.7 2.1215.9 13.7 L180M EC50 >4,000 >1,370 1.8 6.9 366.7 31.5 quadEC90 >435 >263 2.9 5.2 >12,048 >41 AUC 603.4 464.4 1.3 4.2 242.7 21.1V173L EC50 >4,000 >1,370 0.8 1.5 7.0 42.7 triple EC90 >435 >283 0.9 2.979.5 >41 AUC 625.3 1200.0 0.4 2.3 47.5 12.7

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The invention claimed is:
 1. A method of detecting an Hepatitis B Virus(HBV) variant from an HBV isolate, said method comprising: isolating theDNA genome or the corresponding mRNA genome from said HBV isolate;amplifying a portion of the genome, or cDNA from the mRNA, with one ormore biotin-labeled or ligand-labeled primers that selectively bind to aportion of the HBV reverse transcriptase (rt) gene which codes for anaspartic acid (D) at rt54, a lysine (K) at rt53, a lysine (K) at rt53, athreonine (T) at rt122, a glutamic acid at rt139, a lysine (K) at rt149,or a valine (V) at rt233, creating labeled amplification products,wherein the one or more primers do not bind to the portion of the HBV rtgene if the HBV rt gene does not encode for an aspartic acid (D) atrt54, a lysine (K) at rt53, a threonine (T) at rt122, a glutamic acid atrt139, a lysine (K) at rt149, or a valine (V) at rt233, capturing thelabeled amplification products by hybridization with an oligonucleotidethat binds to the biotin or to the ligand, wherein the oligonucleotideis immobilized on a solid support, sequencing the amplificationproducts, and determining whether or not the HBV isolate included anaspartic acid (D) at rt54, a lysine (K) at rt53, a glutamic acid atrt139, a lysine (K) at rt149, or a valine (V) at rt233, wherein theforegoing amino acid positions are numbered in the reference to the M ofthe YMDD motif (Amino Acids 283-286 of SEQ ID No. 10) of the HBV DNApolymerase being position 204 of the HBV DNA polymerase.
 2. The methodof claim 1 wherein the mutation is rtQ149K.